Evaluation of the Efficacy of an Autologous Microbiome Transplant in Adult Atopic Dermatitis Patients
Phase: PHASE1
Conditions: Atopic Dermatitis Eczema
Sponsor: University of California, San Diego

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
Confidential 
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Establishing a Skin Microbiome Transplant  
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Evaluation of the Efficacy of an Autologous Microbiome Transplant in Adult Atopic Dermatitis 
Patients VERSION 2 /May 19, 2016 
NCT03158012 
IND# 15786 
 
 
 
 
 
Study Sponsor(s): The National Institute of Arthritis and Musculoskeletal and Skin Diseases 
(NIAMS) 
Funding Mechanism:   1 R21 AR067547-01A1 
IND Sponsor/Number: IND #15786 
 
 
 
 
 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
Confidential 
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Establishing a Skin Microbiome Transplant  
Version 2 
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INVESTIGATOR SIGNATURE PAGE 
Protocol: 
   
Version/Date:  
 2/ May 19, 2016 
Site Principal Investigator:  Richard Gallo, MD, PhD 
  
Title:    Establishing a Skin Microbiome Transplant 
  
Study Sponsor:   The National Institute of Arthritis and Musculoskeletal and Skin 
Diseases (NIAMS) 
 
INSTRUCTIONS: The site Principal Investigator should print, sign, and date at the indicated 
location below.  A copy should be kept for your records and the original signature page sent. 
After signature, please return the original of this form by surface mail to:  
Richard Gallo, MD, PhD 
9500 Gilman Drive 
Mail Code 0869 
La Jolla, CA 92093-0869 
 
 
I confirm that I have read the above protocol in the latest version.  I understand it, and I will 
work according to the principles of Good Clinical Practice (GCP) as described in the United 
States Code of Federal Regulations (CFR) – 45 CFR part 46 and 21 CFR parts 50, 56, and 312, 
and in the International Conference on Harmonization (ICH) document Guidance for Industry: 
E6 Good Clinical Practice: Consolidated Guidance dated April 1996.  Further, I will conduct 
the study in keeping with local legal and regulatory requirements. 
As the site Principal Investigator, I agree to carry out the study by the criteria written in the 
protocol and understand that no changes can be made to this protocol without the written 
permission of the IRB and NIAMS. 
 
____________________________________     
Site Principal Investigator  (Print) 
 
____________________________________                                      _________________ 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
Confidential 
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Establishing a Skin Microbiome Transplant  
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Protocol Synopsis 
Site Principal Investigator (Signature)                                                          Date 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
Confidential 
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Establishing a Skin Microbiome Transplant  
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Title 
Establishing a Skin Microbiome Transplant 
Short Title 
Establishing a Skin Microbiome Transplant 
Clinical Phase 
1 
Number of Sites 
1 
 
IND Sponsor/Number   
IND 15786 
Study Objectives 
Evaluate the capacity of AMT cream using live bacteria to decrease S. 
aureus colonization in patients with atopic dermatitis (AD). 
 
 
Study Design 
This is a single center phase 1, double-blind placebo controlled trial 
Primary Endpoint(s) 
The change in S. aureus abundance after 7 days of AMT cream using live 
bacteria by using skin swabs to determine the quantity of S. aureus by 
culture and qPCR.  
 
Secondary Endpoint(s)  
Secondary Lesional AD skin:  
1. The abundance of the AMT bacterial strains on lesional skin at 
baseline and after 1 week BID application of AMT.  
2. The quantity of AMT bacterial strains on lesional skin after 
stopping AMT cream 1 day, 2 days, and 4 days after last 
application.  
3. The change in abundance of Staph aureus on lesional skin from 
baseline to 1 day, 2 days, and 4 days after stopping AMT. 
4. Change in clinical assessments, including SCORAD, modified 
EASI, and EASI after 1 week BID application of AMT. 
5. Identify the diversity of the lesional skin microbiome by DNA 
sequencing from baseline and after 1 week BID application of 
AMT. 
6. Evaluate the lesional skin microbiome transcriptome by RNA-
sequencing analysis from baseline and after 1 week BID 
application of AMT. 
Secondary Non-lesional skin:  
1. The abundance of Staph aureus at baseline and after 1 week BID 
application of AMT on non-lesional skin. 
2. The abundance of AMT bacterial strains on non-lesional skin at 
baseline and after 1 week BID application of AMT. 
3. The quantity of AMT bacterial strains on non-lesional skin after 
stopping AMT cream 1 day, 2 days, and 4 days after last 
application.  
4. The change in abundance of Staph aureus on non-lesional skin 
from baseline to 1 day, 2 days, and 4 days after stopping AMT. 
5. Identify the diversity of the non-lesional skin microbiome by 
DNA sequencing from baseline and after 1 week BID application 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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of AMT. 
6. Evaluate the non-lesional skin microbiome transcriptome by 
RNA-sequencing analysis from baseline and after 1 week BID 
application of AMT. 
 
 
1.  
Accrual Objective 
27 subjects  
 
Study Duration 
2 years 
Treatment Description  
Autologous microbiome transplant containing commensal Staph species to 
normalize dysbiosis of atopic dermatitis.  
Inclusion Criteria 
1. Male or female subjects who are not pregnant or lactating 
2. 18-60 years of age 
3. Diagnosis of atopic dermatitis for at least 6 months using the 
Hanifin and Rajka Diagnostic Criteria for atopic dermatitis 
4. Presence of lesional atopic dermatitis skin in both antecubital 
fossae 
5. Positive S. aureus colonization based on results of a skin culture 
taken from one of their AD- affected antecubital fossae during the 
screening visit 
6. Positive for antimicrobial CoNS species from non-lesional AD 
skin 
 
Exclusion Criteria 
1. Use of any topical AD treatments (including topical steroids, 
topical calcineurin inhibitors) to either arm within one week of 
the Treatment visit 
2. Use of any antihistamines 7 days within one week of the 
Treatment visit 
3. Use of any oral/systemic AD therapies (steroids) within 28 days 
of the Treatment visit 
4. Severe AD that would worsen significantly from holding a 
participant’s usual topical/oral AD medications for the time 
periods required in the inclusion/exclusion criteria (one week 
prior to the  Treatment visit for topical medications and 
antihistamines and 28 days prior to  Treatment visits for oral 
medications) 
5. Subjects who have taken a bleach bath within a week prior to the 
Treatment visit, or who take bleach baths during the study 
6. Patients with severe medical condition(s) that in the view of the 
investigator prohibits participation in the study 
7. Subjects with Netherton’s syndrome or other genodermatoses that 
result in a defective epidermal barrier 
8. Any subject who is immunocompromised (e.g. provides 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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researchers with a history lymphoma, HIV/AIDS, Wiskott-
Aldrich Syndrome) or has a history of malignant disease (with the 
exception of non-melanomatous skin cancer).  This information 
will be gathered verbally from the patient while taking a medical 
history from the patient, and will not involve further testing such 
as an HIV test. 
9. Subjects with a history of psychiatric disease or history of alcohol 
or drug abuse that would interfere with the ability to comply with 
the study protocol 
10. Active bacterial, viral or fungal skin infections  
11. Any noticeable breaks or cracks in the skin on either arm, 
including severely excoriated skin or skin with open or weeping 
wounds suggestive of an active infection or increased 
susceptibility to infection. 
12. Ongoing participation in another investigational trial 
13. Use of any oral or topical antibiotic for up to four weeks prior to 
screening 
14. Use of any systemic immunosuppressive therapy (e.g. CsA, 
MTX, etc.) within four weeks of screening. 
15. Sensitivity to or difficulty tolerating Dove fragrance-free bar 
soap, Cetaphil lotion 
16. Subjects with prosthetic heart valves, pacemakers, intravascular 
catheters, or other foreign or prosthetic devices. 
 
Study Stopping Rules 
The following criteria will be used to determine whether or not the study 
should be stopped to protect the interest of all participants: 
1. Any serious adverse event for which there is a reasonable 
possibility that the study product caused the serious adverse 
event. 
2. The development of a serious adverse event for which attribution 
cannot be assessed as definitely unrelated to the study product in 
10% or more of the study population. 
3. The development of any severe (Grade 3) adverse events in 10% 
or more of the study population. 
  
 
 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
Confidential 
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Study Contacts 
 
 
 
 
 
 
 
 
SITE PRINCIPAL INVESTIGATOR 
Richard Gallo, MD, PhD 
Professor of Dermatology 
UCSD 
9500 Gilman Drive 
La Jolla, CA 92093-0869 
Phone: 858-822-4608 
Fax:  858-822-6985 
Email: rgallo@ucsd.edu 
 
 
 
SITE  PRINCIPAL INVESTIGATOR 
Tissa Hata, MD 
Professor of Dermatology 
UCSD 
8899 University Center Lane,Suite 
350 
San Diego, CA 92122 
Phone: 858-657-8390 
Fax: 858-657-8391 
Email:thata@ucsd.edu 
SITE   PRINCIPAL  INVESTIGATOR 
Karsten Zengler, PhD 
Research Scientist 
UCSD 
9500 Gilman Drive 
La Jolla, CA 92093-0412 
Phone: 858-534-5758 
Fax: 
Email: kzengler@ucsd.edu 
 
 
 
CO-INVESTIGATOR 
Teruaki Nakatsuji 
Assistant Project Scientist 
UCSD 
9500 Gilman Drive 
La Jolla, CA 92093-0869 
Phone: 858-822-3955 
Fax: 858-822-6985 
Email: tnakatsuji@ucsd.edu 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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Table of Contents 
Glossary of Abbreviations .................................................................................................................................................... 11 
1. 
Background and Rationale ............................................................................................................................................ 12 
1.1. 
Background and Scientific Rationale .................................................................................................................... 12 
1.2. 
Rationale for Selection of Investigational Product or Intervention ....................................................................... 12 
1.3. 
Preclinical Experience ........................................................................................................................................... 13 
1.4 Clinical Studies ............................................................................................................................................................... 14 
2. 
Study Hypotheses/Objectives ....................................................................................................................................... 16 
3. 
Study Design ................................................................................................................................................................. 17 
3.1. 
Description of Study Design ................................................................................................................................. 17 
3.2. 
Stratification, Randomization, and Blinding/Masking .......................................................................................... 18 
3.2.1. 
Procedure for Unblinding/Unmasking .............................................................................................................. 18 
4. 
Selection of Participants and Clinical Sites/Laboratories ............................................................................................. 19 
4.1. 
Rationale for Study Population ............................................................................................................................. 19 
4.2. 
Inclusion Criteria................................................................................................................................................... 19 
4.3. 
Exclusion Criteria ................................................................................................................................................. 19 
5. 
Known and Potential Risks and Benefits to Participants .............................................................................................. 20 
5.1. 
Risks of Investigational Product ........................................................................................................................... 20 
5.2. 
Risks of Study Procedures .................................................................................................................................... 21 
5.3. 
Potential Benefits .................................................................................................................................................. 21 
6. 
Investigational Agent /Device/Intervention .................................................................................................................. 21 
6.1. 
Investigational Agents/Devices/Interventions ...................................................................................................... 21 
6.1.1. 
Investigational Agent .................................................................................................................................... 21 
6.1.1.1. 
Dosage, Preparation, and Administration ................................................................................................. 21 
6.2. 
Study Design – dose schedule ............................................................................................................................... 22 
6.3. 
Drug Accountability .............................................................................................................................................. 23 
6.4. 
Assessment of Participant Compliance with Investigational Agent ..................................................................... 23 
6.5. 
Toxicity Prevention and Management .................................................................................................................. 23 
6.6. 
Premature Discontinuation of Investigational Agent ............................................................................................ 23 
7. 
Other Medications ......................................................................................................................................................... 24 
7.1. 
Concomitant Medications ..................................................................................................................................... 24 
7.2. 
Prohibited Medications ......................................................................................................................................... 24 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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7.3. 
Rescue Medications .............................................................................................................................................. 24 
8. 
Study Procedures........................................................................................................................................................... 24 
8.1. 
Enrollment ............................................................................................................................................................. 24 
8.2. 
Screening/Baseline Visit ....................................................................................................................................... 24 
8.3. 
Study Visits or Study Assessments ....................................................................................................................... 25 
8.4. 
Unscheduled Visits ............................................................................................................................................... 31 
8.5. 
Visit Windows ...................................................................................................................................................... 31 
9.1 Skin swabs Samples ........................................................................................................................................................ 33 
9.1.1. Skin Swab collection ............................................................................................................................................... 33 
9.1.2. 
Staphylococcus aureus Screening assay ........................................................................................................ 33 
9.1.3. 
9.1.2. CFU Quantification of Live Staphylococcus aureus ........................................................................... 33 
9.1.4. Quantification of Staphylococcus aureus, S. hominis, S. epidermidis, and total Staphylococcus DNA by q PCR
....................................................................................................................................................................................... 33 
10. 
Biospecimen Storage................................................................................................................................................. 34 
11. 
Criteria for Participant and Study Completion and Premature Study Termination .................................................. 35 
11.1. 
Participant Completion ..................................................................................................................................... 35 
11.2. 
Participant Stopping Rules and Withdrawal Criteria ........................................................................................ 35 
11.3. 
Participant Replacement ................................................................................................................................... 35 
11.4. 
Follow-up after Early Study Withdrawal .......................................................................................................... 35 
12. 
Safety Monitoring and Reporting ............................................................................................................................. 36 
12.2 Definitions ................................................................................................................................................................. 36 
12.2.1 Adverse Event (AE) ........................................................................................................................................... 38 
12.3 Grading and Attribution of Adverse Events.............................................................................................................. 39 
12.3.1 Grading Criteria ................................................................................................................................................. 39 
12.3.2 Attribution Definitions ....................................................................................................................................... 40 
12.5 Reporting of Serious Adverse Events and Adverse Events ...................................................................................... 40 
12.6   
Pregnancy Reporting ......................................................................................................................................... 41 
12.8 Review of Safety Information ................................................................................................................................... 41 
12.8.1 Medical Monitor Review ................................................................................................................................... 41 
12.8.2 Safety Officer Review ........................................................................................................................................ 41 
13. 
Statistical Considerations and Analytical Plan ......................................................................................................... 42 
13.1 
Overview ............................................................................................................................................................... 42 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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13.2 
Endpoints/Outcomes ............................................................................................................................................. 42 
13.2.1 Primary Endpoint(s)/Outcome(s) ........................................................................................................................... 43 
13.2.2 Secondary Endpoint(s)/Outcome(s) ....................................................................................................................... 43 
13.4 
Analysis Plan ........................................................................................................................................................ 43 
13.4.1 
Analysis Populations. .................................................................................................................................... 43 
13.4.2 
Primary Analysis of Primary Endpoint(s)/Outcome(s) ................................................................................. 44 
13.4.4 
Analyses of Secondary and Other Endpoint(s)/Outcome(s) ......................................................................... 44 
13.4.5 
Analyses of Exploratory Endpoint(s)/Outcome(s) ........................................................................................ 46 
13.4.6 
Descriptive Analyses ..................................................................................................................................... 46 
13.5 
Interim Analyses ................................................................................................................................................... 47 
13.7 
Sample Size Considerations .................................................................................................................................. 47 
14. 
Identification and Access to Source Data ................................................................................................................. 47 
14.1. 
Source Data ....................................................................................................................................................... 47 
14.2. 
Access to Source Data ....................................................................................................................................... 47 
15. 
Protocol Deviations ................................................................................................................................................... 48 
15.1. 
Protocol Deviation Definitions ......................................................................................................................... 48 
15.2. 
Reporting and Managing Protocol Deviations .................................................................................................. 48 
16. 
Ethical Considerations and Compliance with Good Clinical Practice ...................................................................... 48 
16.1. 
Statement of Compliance .................................................................................................................................. 48 
16.2. 
Informed Consent Process................................................................................................................................. 48 
16.3. 
Privacy and Confidentiality............................................................................................................................... 49 
17. 
Publication Policy ..................................................................................................................................................... 49 
18. 
References ................................................................................................................................................................. 49 
 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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Glossary of Abbreviations 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CTCAE 
Common Terminology Criteria for Adverse Events 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonization 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
MOP 
Manual of Procedures 
PI 
[Site] Principal Investigator 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAR 
Suspected Adverse Reaction 
SOP 
Standard Operating Procedure 
SUSAR 
Serious Unexpected Suspected Adverse Reaction 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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1. Background and Rationale  
1.1. Background and Scientific Rationale 
 It is essential for microbial communities to reside on our epithelial surfaces to maintain several functions of 
human health (1-3).   This conclusion is based in part on observations that dysbiosis, a state of microbial 
imbalance, is associated with various diseases (1). Systemic antibiotics, bleach baths, or other antimicrobial 
interventions have been used in patients with Atopic Dermatitis (AD) to attempt to correct dysbiosis (4). 
Unfortunately, in addition to promoting the development of antibiotic resistance in pathogens, the nonspecific 
use of antimicrobials also further disrupts the microbial ecosystem by killing both beneficial bacteria and 
pathogens. Therefore, the non-specific use of antimicrobials should be avoided when possible.  
 
In this proposal, we describe a novel approach to attempt to treat dysbiosis of the skin surface. We have 
discovered that many of the strains of coagulase negative Staphylococcus (CoNS) that colonize healthy skin, 
such as Staphylococcus epidermidis (S. epidermidis), Staphylococcus hominis, Staphylococcus warneri and 
Staphylococcus capitis, produce antimicrobial activity against Staphylococcus aureus (SA). This activity is 
selective and kills many pathogens but does not inhibit the growth of the normal resident species. AD subjects 
have a deficiency in the abundance of bacteria that produce this activity. We hypothesize that reapplying the 
host’s own antimicrobial bacteria will facilitate elimination of SA colonization.  
 
We propose here to exploit the antimicrobial function of the microbiome as therapy of skin disease. We will 
apply skin commensal microbes that have been pre-identified to have antibacterial (anti-S. aureus) activity. 
These beneficial bacteria will be selected from the subject’s own resident skin flora (autologous) and expanded 
in vitro for reapplication. We wish to determine if this approach will eliminate or reduce S. aureus, and how 
long this will last. Thus, the immediate significance of our work will be to show how the microbiome can be 
used to inhibit SA colonization on the skin. However, the long-term significance of this work will be that it will 
set the groundwork to test if such a transplant of skin commensal bacteria can be of lasting clinical benefit to 
patients with AD. This work will therefore also examine key variables to enable later tests of the capacity to 
alter the microbiome over a longer time period. If successful, the current project will permit future test of 
functions of the skin microbiome such as immune regulation and barrier development (5-9). 
 
1.2. Rationale for Selection of Investigational Product or Intervention 
Unlike healthy control skin, the skin of patients with atopic dermatitis (AD) is frequently colonized by 
Staphylococcus aureus (S. aureus) (Leyden JJ et al. 1974), putting these patients at increased risk of S. aureus 
skin infections.  In addition, research in our lab has shown that these patients have fewer protective 
Staphylococcal species such as Staphylococcal epidermidis (S. epidermidis) that are known to produce 
antimicrobial peptides that play a role in protecting the skin from invading pathogens.  In this study, we will 
attempt to decrease S. aureus colonization and increase colonization by protective Staph species in AD patients 
by first culturing the bacteria on subjects’ lesional AD skin.  We will selectively grow the subject’s S. 
epidermidis colonies and place them into Cetaphil moisturizer.  This will be a single site, double-blind, 
randomized phase 1 clinical trial evaluating a novel therapy for atopic dermatitis, an autologous microbiome 
transplant (AMT).  
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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1.3. Preclinical Experience 
This proposal introduces the innovative concept of autologous microbial transplant (AMT) therapy. The 
principle of skin AMT therapy is to isolate a subset of the subject’s own microbes with beneficial functions, 
expand them in vitro, and then to transplant these specific microbes back onto diseased sites of the same 
subject.  We have developed a novel high-throughput screening technique to select these microbes from skin 
flora. This therapy can be an attractive alternative to antimicrobial therapy because we use bacteria already on 
the subject and these microbes have therefore already demonstrated the capacity to be tolerated by the subject 
and not to cause disease. This theoretically reduces the risk compared to non-native species/strains. Thus, we 
believe the concept of AMT is attractive and innovative.   
Our group’s prior research has focused on the underlying molecular mechanisms for the microbial symbiosis 
that benefits our cutaneous immune system and homeostasis. In a 2009 publication in Nature Medicine, our 
group first demonstrated an anti-inflammatory benefit of S. epidermidis, a predominant CoNS species on 
normal human skin(6). We have also demonstrated that a small molecule of <10 kDa secreted from S. 
epidermidis increased expression of ß-defensins in murine skin and human keratinocytes, enhancing resistance 
to pathogens of mice at a steady state (5).  Other groups also have shown that a product secreted from S. 
epidermidis strongly sensitizes the host’s innate immune system to pathogens (7). In cultured human 
keratinocytes, activation of TLR2 by peptidoglycan, the major constituent of gram-positive cell walls, 
strengthens cellular tight junctions (9). More recently, it was demonstrated that skin commensal bacteria 
enhanced cutaneous T-cell functions via activation of IL-1 signaling (8). Indeed, mono-colonization of germ-
free mouse skin by S. epidermidis protected these mice against pathogen infection (8).   
Some strains of S. epidermidis, often the dominant bacteria found in the skin microflora, produce various types 
of antimicrobials (10). Because of their potential to kill pathogens in vitro, these antimicrobials can provide 
antimicrobial protection against pathogens on the skin surface. We have published several examples of this (5, 
11), and are characterizing more by using our innovative screening technique. These findings indicate that 
multiple CoNS species inhabiting the surface of normal human skin can be a source of antimicrobial activity 
and contribute directly to the skin’s antimicrobial defenses. Also, this shows an additional benefit that may be 
derived from this study. By isolating diverse clones of antimicrobial bacteria we may discover several new 
antimicrobials 
Figure 1 shows the high-throughput technique for screening CoNS strains that produce antimicrobial activity 
against SA. This technique enables us to determine the frequency of antimicrobial CoNS strains on the skin and 
then select them for transplant. Preliminary data shown in our introduction to the revised protocol has illustrated 
how AD patients colonized by SA have a lower frequency of antimicrobial CoNS species than normal healthy 
skin or S. aureus-negative AD patients. The profile of these antimicrobial CoNS was determined to be different 
from that of non-antimicrobial species by sequencing of the full-length 16S rRNA gene of each clone. These 
findings show that the human skin harbors diverse Staphylococcal strains providing antimicrobial activity, and 
that a low frequency of antimicrobial CoNS may partially account for the susceptibility of AD skin to infection 
by SA. All subjects studied to date harbor CoNS species that will inhibit SA. Thus, a source of microbes for 
AMT is available on most subjects even though AD subjects have fewer available with this activity than normal 
subjects. 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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Figure. 1. High-throughput screening 
for antimicrobial activity produced by 
skin commensal staphylococcus 
species.  Coagulase-negative 
Staphylococcus species are isolated on 
mannitol salt agar from skin by 
swabbing. Antimicrobial activity in 
conditioned media from each 
Staphylococcus clone is tested against S. 
aureus 113 strain. 
 
Preliminary work has identified an excellent vehicle for AMT. Clinical isolates of antimicrobial strains of S. 
hominis or known antimicrobial strains of S. epidermidis (ATCC12228) (12) were mixed with several different 
over-the-counter skin moisturizer creams at 1×107 CFU/gm. Cetaphil® Moisturizing Lotion (Galderma, TX) 
was found to be an excellent bacteriostatic vehicle.  This formulation retained the viability of antimicrobial 
Staphylococcus for over 5 days at room temperature, but did not allow bacteria to grow (100% at day-2 and 
50% at day-5).  This formulation was active. A single topical application of this cream formulation containing a 
known antimicrobial strain of S. epidermidis (ATCC12228), or newly discovered antimicrobial strain of S. 
hominis, were found to successfully decrease S. aureus colonization on the skin of mice within 29 hrs in 
comparison to vehicle or similar formulations of known non-antimicrobial S. epidermidis strain (ATCC1457) 
(Fig.2).  These data show that AMT on humans is likely to inhibit S. aureus colonization.   
 
Figure 2. Effect of a bacterial transplant on the survival of 
S. aureus on mouse skin.  Shaved back skin was inoculated 
with 1×105 CFU S. aureus 113 on a 2×2cm area. After 3 hours, 
Cetaphil® cream containing indicated dose of an antimicrobial 
strain of S. hominis (SH) (50mg) or a known antimicrobial 
strain of S. epidermidis (ATCC12228) (SE12228) was topically 
applied. Cetaphil® without bacteria was used as a vehicle 
control, and a non-antimicrobial strain of S. epidermidis 
(ATCC1457) (SE1457) was used as a negative control. S. 
aureus colonies measured 29 hrs later and by counting on a mannitol salt agar plate.  
Clinical Studies 
N/A 
1.4 Clinical Studies 
In order to further examine if topical application of antimicrobial CoNS can decrease S. aureus survival on the 
skin of AD subjects, we conducted a preliminary double-blind, placebo-controlled autologous microbiome 
transplant  study (AMT) under IND# 15786. An antimicrobial CoNS strain was isolated from the non-lesional 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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skin of each of 5 AD subjects who were S. aureus culture positive. To transiently increase their abundance on 
each subject, the active strains were expanded and then reapplied to the lesional forearm skin of the same 
subject, from which the active colonies were isolated, to a final concentration of 1×105 CFU/cm2, a density 
similar to previous assessments of the abundance of bacteria on normal human skin. A single application of this 
functionally defined and autologously-derived CoNS strain(s) decreased S. aureus CFU within 24 hours; 
whereas, vehicle applied to the contralateral arm in a double-blind comparison did not (Fig. 4a-b).  However, 
CoNS survival on the arm 24 hours after AMT was similar to the baseline.  These results suggest that a single 
application of CoNS at a dose of 1×105 CFU/cm2 was sufficient to transiently decrease survival of S. aureus, 
but the majority of transplanted CoNS died within 24 hours.  Therefore, we propose to apply the AMT cream 
for multiple applications (BID for seven days) for better survival of transplanted CoNS.   
 
Figure 4. Transplantation of antimicrobial CoNS reduces survival of S. aureus colonized on the skin surface. 
Figure 4 panel (a) demonstrates the effect of transplantation of antimicrobial CoNS strain(s) on survival of S. 
aureus on the skin surface of subjects with AD. S. aureus survival was measured on the lesional sites in both 
antecubital fossa of the AD subject before and 24 hours after either treatment with antimicrobial CoNS strain(s) 
autologously screened from the same subject or vehicle. Panel (b) illustrates S. aureus survival on the forearm 
24 hours after treatment with autologous strain(s) of CoNS or treatment with vehicle.   Relative CFU of S. 
aureus is shown as percent of baseline and different color symbols indicates data from each individual subject. 
In panel (c) CFUs of CoNS on the skin surface of subjects with AD were simultaneously counted as shown in 
panel (a).  Panel (d) shows CoNS survival on the forearm 24 hours after treatment with autologous strain(s) of 
CoNS or treatment with vehicle.  Relative CFU of CoNS is shown as percent of baseline. 
In our pilot study of 5 subjects evaluating an AMT cream (IND#15786), we noted no serious adverse events 
(SAEs). Two subjects noted mild worsening of AD, which were deemed unrelated to the AMT cream given 
AMT cream was not applied on the area with worsening AD. One subject noted mild worsening of AD with an 
increase in pruritus and erythema on both arms. Since AMT was applied to only one arm, it is less likely that 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
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the adverse event (AE) was related to the AMT cream given the bilateral worsening of AD on the upper 
extremities.  Overall, the AMT cream was very well tolerated and had no significant adverse events. 
 
2. Study Hypotheses/Objectives 
Our preliminary data strongly suggest that some strains of resident CoNS act to defend the skin surface against 
colonization by SA. The goal of this proposal is to restore this function of the microbiome on atopic skin by 
replacing these beneficial bacteria. Our approach will be very safe since we will select the beneficial microbes 
that are normal residents of healthy skin. These bacteria will be harvested from the subject, expanded and 
reapplied to the same subject. An FDA IND has been approved and institutional IRB obtained for this approach 
(IND #15786). After application of the bacteria the survival of SA will be measured. Thus, this autologous 
microbiome transplant (AMT) is a first step before a generalized “probiotic” therapeutic approach.  
We propose here a randomized, double-blind, placebo-controlled trial that will apply beneficial bacteria from 
the AD host onto lesional skin of AD patients, thus performing an autologous microbiome transplant. The 
transplanted bacteria have been selected based on their capacity to inhibit S. aureus colonization. Our central 
hypothesis is that this intervention will decrease colonization of S. aureus and normalize immune functions of 
AD skin. This hypothesis will allow us to evaluate the function of the microbiome in AD, and normalize host 
defense properties in AD, and to develop an intervention that is highly likely to decrease S. aureus colonization 
and may also improve skin inflammation. During the course of this trial we will also obtain vital information to 
further explain how the microbiome functions and what variables control its composition. 
 
Aim Evaluate the capacity of AMT cream to decrease S. aureus colonization in patients with atopic dermatitis 
(AD). 
Hypothesis: Transplant of AMT will decrease S. aureus on AD skin 
Study objectives: 
Primary: To determine the change in abundance of Staph aureus from baseline after 1 week 
BID application of AMT. 
 
Secondary Lesional AD skin: 
1. To measure the abundance of the AMT bacterial strains on lesional skin after 1 week BID 
application of transplant.  
2. To measure the rate of elimination of the microbiome transplant on lesional skin after 
stopping application of transplant cream 
3. To determine the rate of reappearance of Staphylococcus aureus on lesional skin after 
discontinuing 1 week application of AMT cream.  
4. To determine the change in clinical assessments, including SCORAD, modified EASI, and 
EASI after 1 week BID application of AMT.  
5. Evaluate the diversity of lesional skin microbiome by DNA sequencing from baseline and 
after 1 week BID application of AMT.  
6. Measure the difference in microbiome transcriptome on lesional skin by RNA-sequencing 
analysis from baseline and after 1 week BID application of AMT.  
Secondary Non-lesional skin: 
1. To determine the change in abundance of Staph aureus from baseline after 1 week BID 
application of AMT on non-lesional skin. 

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2. To measure the abundance of the AMT bacterial strains on non-lesional skin after 1 week 
BID application of transplant.  
3. To measure the rate of elimination of the microbiome transplant on non-lesional skin after 
stopping application of AMT 
4. To determine the rate of reappearance of Staphylococcus aureus on non-lesional skin after 
discontinuing 1 week application of AMT cream.  
5. Evaluate the diversity of lesional skin microbiome by DNA sequencing from baseline and 
after 1 week BID application of AMT on non-lesional skin.  
6. Measure the difference in microbiome transcriptome on non-lesional skin by RNA-
sequencing analysis from baseline and after 1 week BID application of AMT.  
 
 
3. Study Design 
3.1. Description of Study Design  
This is a single site, double blind placebo controlled study, which will assess the efficacy, safety, and steady 
state of Autologous Microbiome Transplant (AMT) cream in adults with AD. After providing informed consent, 
patients will be assessed for study eligibility at the screening visit, in which participants will consent to all of 
the study experiments separately.  Subjects that withdraw from the study will be replaced. Eligible subjects will 
be randomized to receive either AMT cream or placebo in a 2:1 allocation ratio (AMT cream: placebo). 
Subjects will be instructed to apply either the AMT or vehicle cream to both arms twice a day for seven days. 
Both subjects and clinical investigators will be blinded. Optional follow-up visits to assess clinical effect and 
steady state may occur at 1 and 4 hours after initial cream application if the subjects are willing to return at 
these time points. 4 days after the treatment visit, subjects will return to the clinic for fresh aliquots of the AMT 
and vehicle creams as well as to assess safety and clinical effect. Subjects will return to clinic after the seven 
day course of AMT or vehicle creams for clinical and safety assessments. Subjects will then be asked to return 
to the clinic 1 day after completion of the AMT or vehicle cream course for additional safety and clinical effect 
assessments. Subsequently, subjects will return for additional follow-up visits only if their lesional atopic 
dermatitis sites have not yet returned to baseline (S. aureus is no longer inhibited and Staph species from the 
AMT cream is zero). Up to 27 subjects will be recruited for this entire study. The duration of this studyis 
approximately 2 months. We anticipate the entire study duration will be approximately 2 years total, and that it 
will take approximately 1 year to reach recruitment and enrollment goals for this study. The study flow diagram 
is provided in Figure 1.  
 
 
Intervention(s) and comparators: A coded cream consisting of Cetaphil® Moisturizing Lotion alone, or the 
microbiome transplant consisting of Cetaphil® Moisturizing Lotion plus 105-8 CFU/gram Cetaphil® of 
coagulase negative Staphylococci clones expressing antimicrobial products isolated from the subject’s own non-
lesional skin. Since the two creams are indistinguishable in terms of color and texture, both the study 
coordinator and the subject will remain blinded to the identity of the cream. Our goal is to achieve a density of 1 

Investigator Name: Richard Gallo, MD, PhD 
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X 105 CFU/10mg/cm2 at the time of application. This abundance is known to be safe as it is the density of 
bacteria typically found on normal skin.  
 
 
 
 
[Insert a schematic diagram of the trial here (include study cohorts and sample size, treatment allocation, time 
points of study intervention and subject follow-ups]) 
 
Table format  
Cohort  
27 subjects 
ARM 1 
18 subjects: 
AMT cream  
ARM 2 
9 subjects: 
Vehicle cream 
only 
 
 
3.2. Stratification, Randomization, and Blinding/Masking 
This is a randomized, double-blind, placebo controlled study. After informed consent is obtained, eligibility 
information will be relayed to an unblinded laboratory staff member who will serve as the unblinded 
pharmacist. This staff member will then randomize subjects in a 2:1 allocation ratio (AMT cream: placebo). 
This unblinded pharmacist will use an online tool to generate a simple randomization list: 
http://www.randomization.com/. This lab member will notify the clinical team once participant has been 
randomized and is responsible for securely storing all randomization files.  
3.2.1. Procedure for Unblinding/Unmasking 
In order to minimize potential bias from Investigators, investigational staff, and patients, the study will 
be conducted in a double-blind manner, except for the unblinded laboratory staff for study drug 
preparation, who will be the only unblinded study site personnel at the site. 
 
Unblinding will occur in cases of emergency situations requiring the knowledge of a given treatment. As 
per regulatory reporting requirements, the blind will be broken for all unexpected serious adverse events 
that are considered by the investigator to be related to the study drug. Details of patients who are 
unblinded during the study will be included in the Clinical Study Report. The blind will be broken only 
if specific emergency treatment would be dictated by knowing the treatment status of the patient.  
Unblinding will be approved by the study Safety Officer unless an immediate life threatening condition 
has developed and the Safety Officer is not accessible. The site investigator will also notify KAI and 
NIAMS regarding the emergency unblinding event on the next business day.  

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A full account of the event will be recorded, including the date and time of the unblinding, the reason for 
the decision to unblind, and the name of the individual who made the decision and the names of the 
Medical Monitor and others who were notified. The reasons for unblinding of a participant’s treatment 
will be included in the final study report. 
Unblinding the study due to an approved interim analysis, final analysis, or study termination will 
require written approval from NIAMS. 
4. Selection of Participants and Clinical Sites/Laboratories 
4.1. Rationale for Study Population 
In atopic dermatitis, a state of microbial imbalance known as dysbiosis occurs and is closely associated with 
disease severity. An important characteristic of the dysbiosis in AD is an increased abundance of S. aureus and 
a decrease in overall bacterial diversity. Several lines of experimental evidence support the concept that the 
multiple defects in epithelial barrier function in AD promote dysbiosis and that this dysbiosis then promotes the 
immunological disorder characteristic of AD. 
 
 
 
4.2. Inclusion Criteria  
Up to 54 subjects with atopic dermatitis will be enrolled in this study. Enrollment will not take race or gender 
into consideration.  Pregnant women, children (under age 18), and unhealthy subjects with significant co-morbid 
conditions as determined by the PI will be excluded. 
1. Male or female subjects who are not pregnant or lactating.  Female subjects of child-bearing 
potential must have a negative urine pregnancy test on the day of the screening visit in order to be 
eligible for the study. 
2. 18-60 years of age 
3. Diagnosis of atopic dermatitis for at least 6 months using the Hanifin and Rajka Diagnostic Criteria 
for atopic dermatitis 
4. Presence of lesional atopic dermatitis skin in both antecubital fossae 
5. Positive S. aureus colonization based on results of a skin culture taken from one of their AD- 
                  affected antecubital fossae during the screening visit 
6. Positive for antimicrobial CoNS species from non-lesional AD skin 
4.3. Exclusion Criteria 
Individuals who meet any of these criteria are not eligible for enrollment as study participants:  
1. Use of any topical AD treatments (including topical steroids, topical calcineurin inhibitors) to either 
arm within one week of  the Treatment visit 
2. Use of any antihistamines 7 days within one week of the Treatment visit 
3. Use of any oral/systemic AD therapies (steroids) within 28 days of the Treatment visit  
4. Severe AD that would worsen significantly from holding a participant’s usual topical/oral AD 
medications for the time periods required in the inclusion/exclusion criteria (one week prior to the  

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Treatment visit for topical medications and antihistamines and 28 days prior to  Treatment visits for 
oral medications) 
5. Subjects who have taken a bleach bath within a week prior to the Treatment visit, or who take bleach 
baths during the study 
6. Patients with severe medical condition(s) that in the view of the investigator prohibits participation 
in the study 
7. Subjects with Netherton’s syndrome or other genodermatoses that result in a defective epidermal 
barrier 
8. Any subject who is immunocompromised (e.g. provides researchers with a history lymphoma, 
HIV/AIDS, Wiskott-Aldrich Syndrome) or has a history of malignant disease (with the exception of 
non-melanomatous skin cancer).  This information will be gathered verbally from the patient while 
taking a medical history from the patient, and will not involve further testing such as an HIV test. 
9. Subjects with a history of psychiatric disease or history of alcohol or drug abuse that would interfere 
with the ability to comply with the study protocol 
10. Active bacterial, viral or fungal skin infections  
11. Any noticeable breaks or cracks in the skin on either arm, including severely excoriated skin or skin 
with open or weeping wounds suggestive of an active infection or increased susceptibility to 
infection. 
12. Ongoing participation in another investigational trial 
13. Use of any oral or topical antibiotic for up to four weeks prior to the Treatment visit 
14. Use of any systemic immunosuppressive therapy (e.g. CsA, MTX, etc.) within four weeks of the 
Treatment visit. 
15. Sensitivity to or difficulty tolerating Dove fragrance-free bar soap or Cetaphil lotion 
16. Subjects with prosthetic heart valves, pacemakers, intravascular catheters, or other foreign or 
prosthetic devices. 
 
5. Known and Potential Risks and Benefits to Participants 
5.1. Risks of Investigational Product  
Risk of applying microbial cream (Cetaphil® containing Staphylococcal bacteria) 
Skin infection 
The likelihood of subjects developing a skin infection is low given that we will be transplanting their non-
pathogenic autologous bacteria that have antimicrobial peptide-producing abilities.  Since we are 
transplanting the subject’s own endogenous bacteria back onto them, we know that these bacteria are 
present on the subject’s skin at baseline without causing an infection.  In addition, the bacteria strains to be 
transplanted onto the patient will be screened prior to putting it into the AMT cream to ensure that it is not a 
skin pathogen.  The known pathogen S. aureus that frequently colonizes AD skin will not even be 
considered as a potential bacteria to transplant because of its yellow color on mannitol-salt-agar plates, 
which isolated bacteria will be grown on.  Only red colonies (S. epidermidis) growing on mannitol-salt-agar 
plates will be chosen and screened as potential bacteria to be transplanted onto the subject.  If anything, we 
expect the population of S. aureus to be reduced with the use of the AMT cream, since the AMT cream will 
contain only bacteria that are known to inhibit S. aureus.  Furthermore, we will be applying these bacteria 
topically and will exam all patients first to ensure that they do not have any cracks or excoriated skin on 
their arms before we apply it.  We will also avoid applying the microbial cream to the hand since the hand 
would be a likely source of spreading these bacteria onto other surfaces that other people may contact.  
Should an infection occur (or should the subject be concerned about an infection), the subject should call 
Dr. Tissa Hata or Dr. Richard Gallo.  The subject would be treated according to the standard treatment after 
examination by Drs. Tissa Hata or Richard Gallo. The treatment will include topical and/or oral antibiotics 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
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(for infection) depending on the severity of the infection clinically, and Cetaphil® moisturizing lotion (for 
irritation and dryness), provided by Drs. Tissa Hata or Richard Gallo.  The topical antibiotic of choice will 
be mupirocin.  The oral antibiotic of choice will depend on the individual subject’s medical history, 
including any possible contra-indications to certain medications due to allergies or other concomitant 
medication use, but doxycycline will be the first line oral antibiotic, followed by a cephalosporin or 
penicillinase-resistant penicillin. 
If the subject calls to report a possible infection during a time when the clinic is closed, the subject will be 
directed to the closest urgent care facility for further management. 
5.2. Risks of Study Procedures  
Risk of loss of confidentiality:  Every reasonable effort will be made to keep records confidential.  Research 
records will be stored in a secure building under lock and key of the study personnel.  Data stored in our 
computers are password protected.   Unless required by law, only the Study Doctors, the Study Team, the 
UCSD Institutional Review Board, the Food and Drug Administration, and the National Institutes of Health 
(NIH) will have access to confidential data which identifies you by name. Because of the need to give these 
parties access to this information, absolute confidentiality cannot be guaranteed. 
 
 
Withdrawal from Medications 
Adverse events associated with stopping the use of protocol-prohibited medications may result in worsening of 
the condition being treated and reported as such. As listed in the inclusion/exclusion criteria, subjects with 
severe AD who may have difficulty tolerating periods without medication use will be excluded from 
participating. 
Skin Swabs 
There are no significant risks associated with taking swabs of the skin. 
 
5.3. Potential Benefits 
There are no direct benefits for the subjects who elect to enroll in this study. The results may provide 
identification and/or validation of new targets for the future development of therapeutics for subjects with 
atopic dermatitis, as well as increase current knowledge on the ability to transform the cutaneous microbiome, 
which could lead to potential therapeutic strategies for treating a variety of inflammatory skin conditions. 
6. Investigational Agent /Device/Intervention  
6.1. Investigational Agents/Devices/Interventions 
6.1.1. Investigational Agent  
6.1.1.1. Dosage, Preparation, and Administration 
After isolation of each subject’s antimicrobial Staphylococcus species from their skin swabs, a stock will 
be created that contains equal parts of each of these antimicrobial bacteria.  This stock will be assigned a 
unique batch number, and the contents of this batch are what will be mixed with Cetaphil® at a 

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concentration between 1 x 105 and 1 x 108 CFU/gram Cetaphil® to create the subject’s final AMT cream.  
This stock will be stored at -80C until the time when that subject’s AMT cream is to be formulated.  Prior 
to formulating the final AMT cream product that will be applied on the skin of that subject, though, this 
stock will undergo Microbial Limits Testing in accordance with USP <62>, as well as Microbial Limits 
Testing for the detection of Streptococcus pyogenes, Acinetobacter baumanni, and the skin fungus 
trichophyton in our laboratory both before and after formulation of the AMT cream with Cetaphil®.   
The stock will be stored at -80C to prevent bacterial growth in the stock solution itself.  If the stock is 
free from contaminants as shown by USP <62> testing, the stock will be formulated with Cetaphil® under 
sterile conditions in a sterile hood to prevent contamination. The stock will then be returned to -80C.  If 
the stock is NOT free from contaminants as shown by USP <62> testing, the stock will be discarded 
appropriately.  A sample of formulated AMT cream will then undergo UPS <62> tests for the organisms 
noted previously.  Formulation of the subject’s AMT cream will occur under sterile conditions in a sterile 
laminar flow hood to prevent contamination of the stock and AMT cream.  A sample of Cetaphil® from 
each container of Cetaphil® used for the formulation of AMT cream will also undergo USP <62> testing 
within a week of using that Cetaphil® container for AMT cream formulation. 
Bacterial concentration of the AMT cream will then be checked by plating a known quantity of the AMT 
cream on an agar plate and incubating the plate overnight at 37 ̊ C. The number of viable colonies will be 
counted the following day. Only AMT that has a final concentration of bacteria that ranges from 1 x 105 
CFU of bacteria/ gram Cetaphil® to 1 x 108 CFU of bacteria/ gram Cetaphil® will be used in the clinical 
trial. 
The AMT cream will be provided in single-dose sealed containers. The cream should be stored at room 
temperature per USP definition of “controlled room temperature” for up to one week. The AMT cream is 
meant to be applied topically two times a day at a dose of 3 x 107 CFU/ gram Cetaphil®. 
 
6.2. Study Design – dose schedule 
The dosing schedule for the AMT cream or the Vehicle (Cetaphil® lotion) is described in Sections 8.3 and 8.5.  
The first dose of the AMT cream or Vehicle cream will be administered to the subjects at the clinical site on 
Day 0 (Treatment Initiation Visit). At that time, they will be guided and given a handout instructing them to 
apply the AMT or Vehicle cream uniformly across both arms (this area will include S. aureus colonized lesions 
as well as non-lesional skin) twice daily for seven days. The participants will also be provided a subject diary 
and 18 single-use containers for dosing at home. An additional 14 single-use packets of AMT Product or 
Vehicle Product will be dispensed at the next clinic visit on Day 4 to use until Day 7. Subjects will be instructed 
that 2 additional single-use containers will be provided to them at Day 0 and Day 4 in the event that the subject 
should misplace a container of study cream. Accountability of the study creams will be performed, and subjects 
will be asked to return the jars of used and unused cream at the next in-clinic visit.  
At the clinic visits on Day 4 and Day 7, the subjects will provide skin swab samples and be assessed for adverse 
events (AEs). Three additional clinic visits will be scheduled to assess safety and stability of the AMT cream. 
There will be a 30 day follow-up of all participants after the last treatment to assess safety and disease status. 
This will be brief phone call, and subjects will be asked to report any new adverse events and to answer 
questions regarding the status of their AD. Any subject with an ongoing AE/SAE at the time of this phone 
contact will continue to be followed until the event is resolved with or without sequelae. 

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6.3. Drug Accountability 
Under Title 21 of the Code of Federal Regulations (21CFR §312.62) the investigator will maintain adequate 
records of the disposition of the investigational agent, including the date and quantity of the drug received, to 
whom the drug was dispensed (participant-by-participant accounting), and a detailed accounting of any drug 
accidentally or deliberately destroyed. 
Records for receipt, storage, use, and disposition will be maintained by the study site.  A drug-dispensing log 
will be kept current for each participant.  This log will contain the identification of each participant and the date, 
quantity of drug dispensed, as well as the batch identifying number. 
All records regarding the disposition of the investigational product will be available for inspection.  Unused 
product will be destroyed as per UCSD’s hazardous waste protocol.   
6.4. Assessment of Participant Compliance with Investigational Agent 
Patients will be required to return all aliquots of medications given, used or unused, and each vial will be 
weighed and weight recorded. 
6.5. Toxicity Prevention and Management 
All steps will be taken to minimize potential risks of the study. Patients will be excluded from study with known 
sensitivities/allergies to any of the products used during this study. Patients with any cracks or breaks in their 
skin, including severely excoriated or bleeding skin suggesting that the patient may be susceptible to an 
infection will be excluded. Similarly, subjects with prosthetic or implanted devices will also be excluded. Any 
adverse event, defined as any undesirable sign, symptom, or medical condition occurring after the subject’s 
written consent to participate is completed, will be recorded and reported to the PI as well as the Institutional 
Review Board as required. If the PI believes an AE is possibly related to the study, the PI will determine 
whether or not it is in the best interest of the patient to continue in the study. To protect patient confidentiality, 
all samples will be encoded and anonymous. Strict adherence to current HIPPA guidelines will minimize the 
risk of loss of confidentiality. If the subject is injured as a result of participation in this research, treatment will 
be available. In the case of a skin infection, the subject would be treated according to standard treatment 
practices. The treatment will include antibiotics (for infection) and ointment (for irritation and dryness). 
 
6.6. Premature Discontinuation of Investigational Agent 
Study therapy may be prematurely discontinued for any participant for any of the following reasons: 
1. Evidence of infection either cutaneously, or systemically. 
2.  Severe worsening of AD which in the opinion of the investigator requires stopping of the medication. 
3.  Evidence of contact allergy to investigative medication. 
4.  Evidence of non-compliance to study protocol that in the opinion of the investigator requires discontinuation 
of investigational agent. 
5.  Study therapy may also be prematurely discontinued for any participant if the investigator believes that the 
study treatment is no longer in the best interest of the participant. 

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7. Other Medications 
7.1. Concomitant Medications 
7.1.1. Protocol-mandated 
At the start of the study, eligible subjects will receive Dove soap as well as Cetaphil moisturizing lotion 
to use throughout the duration of the study.  
 
7.2. Prohibited Medications 
1. Use of any topical AD treatments (including topical steroids and topical calcineurin inhibitors) to either 
arm within one week of the Treatment visit 
2. Use of antihistamines within 7 days of the Treatment Visit 
3. Use of any systemic AD therapies or any systemic immunosuppressive therapy (steroids, CsA, MTX, 
etc.) within 28 days of the Treatment visit 
4. Use of any oral or topical antibiotics for up to four weks prior to the Treatment Visit 
5. Use of bleach baths within 7 days of the Treatment Visit 
7.3. Rescue Medications  
If infection occurs, the treatment will include topical and/or oral antibiotics, depending on the severity of the 
infection clinically, and Cetaphil® moisturizing lotion (for irritation and dryness).  The topical antibiotic of 
choice will be mupirocin.  The oral antibiotic of choice will depend on the individual subject’s medical history, 
including any possible contraindications to certain medications due to allergies or other concomitant medication 
use, but doxycycline will be the first line oral antibiotic, followed by a cephalosporin or penicillinase-resistant 
penicillin. 
8. Study Procedures 
8.1. Enrollment 
The research study will be explained in lay terms to each potential research participant. The potential participant 
will sign an informed consent form before undergoing any study procedures. Once the informed consent has 
been signed, and meets all the inclusion and exclusion criteria, the participant is considered enrolled in the study 
and will be assigned a unique participant number. 
8.2. Screening/Baseline Visit 
The purpose of the screening period is to confirm eligibility to continue in the study.  
1. The following procedures, assessments, and laboratory measures will be conducted to determine 
participant eligibility: Prior to beginning any study procedures, subjects will first be asked to review 
and sign the informed consent form.   
2. After obtaining informed consent, the inclusion/exclusion criteria will be reviewed. 
3. A brief medical history and physical exam/vital signs will be performed to assess whether the 
subject meets the required criteria for the study.   
4. Female subjects of child-bearing potential will complete a urine pregnancy test while in the office to 
ensure that they are not pregnant.  

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5. Patients who meet the inclusion and exclusion criteria will then have their lesional AD skin scored 
according to the localized SCORAD grading scale, and the extent of AD lesional skin on their 
bilateral arms will be recorded.    
6. A skin swab performed on the lesional atopic dermatitis skin of their antecubital fossae, will be 
performed to detect the presence of S. aureus.   
Subjects who do not meet the inclusion/exclusion criteria due to the application or use of prohibited medications 
within the timeframe noted will be considered screen-failures.  Skin swabs of these patients will not be 
performed.  These subjects may return for another screening visit after completing the necessary wash-out 
period (7 days for topical medications, 28 days for oral medications) if they are interested in doing so. Subjects 
eligible for this study will be randomized at the Treatment visit 1 to either AMT cream or placebo in a 2:1 
allocation ratio (AMT cream: placebo).  
 
8.3. Study Visits or Study Assessments  
 
Aim:To determine the change in abundance of Staph aureus from baseline after 1 week BID application of 
AMT. 

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The study flowchart is provided below: 
 
This experiment  will consist of up to 9 office visits (Screening, Treatment, 2 optional visits, and 5 Follow-up 
visits) and two phone calls.  All office visits will occur at the UCSD Dermatology Clinic.  Up to twenty seven 
(27) subjects with AD will be recruited for this study. The exact study procedures are noted below:   
Screening Visit 1 (Day -60 to Day -2) 
Prior to beginning any study procedures, subjects will first be asked to review and sign the informed consent 
form.  After obtaining informed consent, the inclusion/exclusion criteria will be reviewed.  A brief medical 
history, vital signs, and physical exam will be performed in helping to assess whether the subject meets the 
required criteria for the study.  Female subjects of child-bearing potential will complete a urine pregnancy test 
while in our office to ensure that they are not pregnant.  Patients who meet the inclusion and exclusion criteria 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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will then have their lesional AD skin scored according to the SCORAD grading scale, EASI, and modified 
EASI, and localized SCORAD, and the extent of AD lesional skin on their bilateral arms will be recorded.  A 
skin swab will be performed on the lesional atopic dermatitis skin of their antecubital fossae bilaterally to detect 
the presence of S. aureus.  Additionally, we will take four (4) culture swabs from the subject’s non-lesional (no 
AD) skin on their upper arms.  These culture swabs will be placed in tryptic soy broth (TSB) until processed in 
the lab, where they will be used to grow the subject’s antimicrobial peptide-producing Staph species to use for 
the autologous microbiome transplant.  Subjects will then receive a bar of Dove moisturizing soap, which they 
will be instructed to use whenever they shower until after the follow-up visit is complete. This will conclude the 
screening visit.  The total time for this visit will be approximately 40 minutes.   
Subjects who do not meet inclusion and exclusion criteria due to their concomitant medications will be asked 
whether they would be willing to washout of their medications prior to Visit 3 should they have both S. aureus 
and CoNS colonization or for a repeat culture visit (if negative for S. aureus or CoNS).  Subjects who agree to 
the washout will have swabs collected for assessment of S. aureus and CoNS colonization.  Subjects who do not 
wish to washout out of the prohibited medications will be identified as screen failures and will not continue in 
the study. 
 
Visit 2, Phone 1 (Day -59 to Day -1) 
Subjects will be notified via telephone whether or not their skin swab grew S. aureus and whether they have 
antimicrobial Staphylococcus species present.  Subjects with a skin culture positive for S. aureus and 
antimicrobial coagulase-negative Staph (CoNS) will be enrolled in the study and visit 3 (Treatment visit) will be 
scheduled.  For these subjects, they will be reminded to withhold their medications prior to Visit 3 according to 
protocol. Subjects  with a negative S. aureus skin culture and/or are negative for CoNS, will be asked to 
withhold their medications according to the protocol (7 days for AD topical medication and antihistamines, and 
28 days for oral AD medications), and a repeat screening visit will be scheduled for additional skin culture 
swabs .The total phone conversation should take approximately 5 minutes. 
 
 
Repeat culture visit, as necessary (Day -31 to -2)  
The purpose of the Repeat Culture Visit is to repeat skin swabbing to assess for the presence of S. aureus and 
CoNS following their medication washout (28 days for oral AD medications, 7 days for topicals and 
antihistamines). This visit will only be required for subjects who had negative cultures for S. aureus and/or 
CoNS and agree to washout of their medications for repeat swabbing.  
 
Inclusion and exclusion criteria will be reviewed. Interim medical history and a physical exam will be 
performed. Female subjects of child-bearing potential will complete a urine pregnancy test while in our office to 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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ensure that they are not pregnant.  Vital signs will be taken. Two (2) skin culture swabs total will be taken of 
their lesional antecubital fossae to assess for S. aureus. Additionally, four (4) skin culture swabs will be taken of 
their non-lesional skin to assess for antimicrobial CoNS. These culture swabs will be placed in tryptic soy broth 
(TSB) until processed in the lab, where they will be used to grow the subject’s antimicrobial peptide-producing 
Staph species to use for the autologous microbiome transplant.  This will conclude the repeat culture visit.  The 
total time for this visit will be approximately 20 minutes.   
 
Repeat phone call, as necessary (Day -30 to -1) 
Subjects will be notified via telephone whether or not their skin swabs grew S. aureus and whether they have 
antimicrobial Staphylococcus species present.  Subjects with a skin culture positive for S. aureus and 
antimicrobial coagulase-negative Staph (CoNS) will be enrolled in the study. For these subjects, Visit 3 
(Treatment visit) will be scheduled. Subjects  with a negative S. aureus skin culture or are negative for CoNS 
will be identified as screen failures.  
 
 
 
Visit 3, Treatment Visit (Day 0 ) 
Eligible subjects with + S. aureus and CoNS screening cultures will be enrolled in the study and instructed to 
return to the clinic.  This waiting period will allow laboratory staff to grow and prepare the subject’s 
endogenous AMP-producing bacteria to be mixed with Cetaphil for application at the treatment visit.  A 
laboratory staff member will serve as an unblinded pharmacist and once eligibility information is relayed to the 
unblinded pharmacist, the subject will be randomized to an allocation ratio of 2:1 (AMT cream: vehicle cream). 
The subject will be asked not to bathe, shower, or exercise on the day of the treatment visit, and also that the 
application of any topical products to their arms on this day is prohibited.  Hand washing is allowed, but 
subjects will be instructed to take care not to apply soap or water above their wrists.   
 
This visit will begin by reviewing the subject’s interim medical history, including medication history to ensure 
that he/she did not use any topical AD treatments on their arms or antihistamines within 7 days or oral AD 
treatments within 28 days of this visit.  For all subjects, a focused physical exam will then be performed of the 
subject’s bilateral arms to ensure that there is no evidence of cracks, breaks or infections of the skin on the 
arms.  Vital signs will be taken. The localized SCORAD, modified EASI, global EASI and SCORAD grading 
scale will be used to rate the severity of the lesional AD skin on the subject’s bilateral arms, and the extent of 
lesional AD skin on each arm will be recorded.  Digital photographs of the lesional skin on both arms will be 
taken.  Each photograph will also include a ruler so the scale of the lesions can be determined, as well as a card 
with the subject’s identification number, visit date, and photograph location so that the photographs can be 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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identified when uploaded.  Next, up to 3 skin swabs will be performed on each arm in a region of eczematous 
skin and non-lesional skin, for a total of up to 12 swabs. For the skin swabs, one culture swab and one to two 
DNA swabs will be taken from the eczematous skin from each arm, and one culture swab and one to two DNA 
swabs will be taken from the non-lesional skin on each upper inner arm for a total of up to twelve (12) swabs. 
After swabbing, the swabs are placed into either an empty Eppendorf tube (if analyzing the DNA of the bacteria 
present on the skin surface) or into a liquid medium such as tryptic soy broth (if analyzing the amount of 
bacteria present on the swabbed skin surface).   These swabs will be analyzed in the lab to determine how many 
and what type of bacteria are present.  The AMT cream or vehicle cream will then be applied to both arms by 
the gloved hands of a study coordinator.  The moisturizers will be provided by Dr. Gallo’s laboratory and will 
be in identical containers labeled either A or B.  Neither the study coordinator nor the subject will know which 
cream is in which container. Container A or B will contain either Cetaphil alone or Cetaphil plus colonies of the 
subject’s endogenous AMP-producing Staph grown in the lab from the subject’s screening swab.  A thin coat of 
cream A or B will be applied to both arms (excluding hand) in order to demonstrate proper application.  Up to 
twelve (12) skin swabs will then be performed on both arms in the areas of cream application on lesional and 
non-lesional skin, between 5 minutes to an hour after cream application, depending on the discretion of the 
investigator. Following the skin swabs, the Cetaphil alone or Cetaphil with AMP-producing Staph will be 
reapplied onto the subjects’ arms as previously described. The subject will be instructed to withhold use of any 
topical or oral AD medications until after their last in-clinic follow-up visit (Visit 8). The subject will be 
provided with aliquots of either the transplant or vehicle creams, enough for at least 4 days of application. 
Subjects will be instructed to apply the cream twice a day for the next 4 days, and will be instructed to apply the 
provided cream from one aliquot per arm. To minimize cross contamination, we will instruct subjects to wear 
long sleeve clothes and change into clean clothes every day. All creams should be stored at room temperature. 
Subjects will be reminded to use Dove soap provided to them, whenever they shower, but to avoid the upper 
extremities, until after their last in-clinic visit on Day 11 is complete. Subjects will be instructed to continue 
their daily skin care routine, and discouraged from using antibacterial soaps and swimming in the ocean or pool 
until their last in-clinic follow-up visit. They will also be instructed that if these events occur, to please notify 
the study staff at their next visit. At the end of the visit, subjects will be given an instruction sheet and a subject 
diary to assess for cream application appliance. They will be asked to bring the diary pages to their Follow-up 
Visit 4. This will conclude the treatment visit.  We expect this visit to take approximately 20 minutes.  
 
Optional Visit 1 (1 hour after cream application +/- 2 hours): Subjects will have the option to return to clinic 
for an optional visit approximately 1 hour after AMT cream application. At this optional visit, up to 12 skin 
swabs will be taken of the lesional and non-lesional skin on the arms (one culture swab and one to two DNA 
swabs for lesional and non-lesional skin of each arm). This visit will take approximately 15 minutes.  
 
Optional Visit 2 (4 hours after cream application +/- 2 hours): Subjects will have the option to return to 
clinic for an optional visit approximately 4 hours after AMT cream application. At this optional visit, up to 12 
skin swabs will be taken of the lesional arms and non-lesional skin on the arms (one culture swab and one to 
two DNA swabs for lesional and non-lesional skin of each arm). This visit will take approximately 15 minutes.  

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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Visit 4 (Day 4 +/- 5): Subjects will return to the clinic to pick up fresh aliquots of the AMT or vehicle creams, 
and to return approximately 4 hours after last cream application. Any adverse events will be recorded. Subjects 
will be asked to refrain from bathing, exercising, or showering starting at 12:01am of Visit 4.  Vital signs will 
be taken. Subject diaries will be collected at this time and a new subject diary will be dispensed to assess for 
cream application appliance.  Used aliquots of cream will be collected at this visit. The localized SCORAD, 
modified EASI, global EASI and SCORAD grading scale will be used to rate the severity of the lesional AD 
skin on the subject’s bilateral arms, and the extent of lesional AD skin on each arm will be recorded.  Digital 
photographs of the lesional skin on both arms will be taken.  Each photograph will also include a ruler so the 
scale of the lesions can be determined, as well as a card with the subject’s identification number, visit date, and 
photograph location so that the photographs can be identified when uploaded.  Next, one culture swab and one 
to two DNA swabs will be taken from the eczematous skin from each arm, and one culture swab and one to two 
DNA swabs will be taken from the non-lesional skin on each upper inner arm for a total of up to twelve (12) 
swabs. We expect this visit to take approximately 15 minutes. 
 
Visit 5 (Day 7 +/- 7):  Subjects will be asked to return to the clinic for Visit 5 approximately 4 hours after last 
cream application.  Subject diaries will be collected at this time.   Any used and unused aliquots of cream will 
be collected at this visit. Subjects will be asked to refrain from bathing, exercising, or showering starting at 
12:01am of Visit 5.  Vital signs will be taken. Any adverse events since the last visit will be recorded.  A brief 
exam of the skin on subjects’ arms will be performed.  The severity of AD on each arm will be graded using the 
localized SCORAD, modified EASI and global SCORAD and EASI grading.  Subjects will also be asked to 
grade their pruritus on a scale of 1-10, with 10 being the worst pruritus of their life.  Digital photographs of the 
lesional skin on both arms will be taken.  Next, one culture swab and one to two DNA swabs will be taken from 
the eczematous skin from each arm, and one culture swab and one to two DNA swabs will be taken from the 
non-lesional skin on each upper inner arm for a total of up to twelve (12) swabs to determine the microbial 
diversity and abundance.  We expect this visit to take approximately 15 minutes. 
 
Visit 6 (Day 8 +/- 7):  Subjects will be asked to return to the clinic for follow-up. Any adverse events since the 
last visit will be recorded.  A brief exam of the skin on subjects’ arms will be performed.  Subjects will be asked 
to refrain from bathing, exercising, or showering starting at 12:01am of Visit 6.  Vital signs will be taken. The 
severity of AD on each arm will be graded using the localized SCORAD, modified EASI, and global SCORAD 
and EASI grading.  Subjects will also be asked to grade their pruritus on a scale of 1-10, with 10 being the worst 
pruritus of their life.  Digital photographs of the lesional skin on both arms will be taken.  Next, one culture 
swab and one to two DNA swabs will be taken from the eczematous skin from each arm, and one culture swab 
and one to two DNA swabs will be taken from the non-lesional skin on each upper inner arm for a total of up to 
twelve (12) swabs. Any used cream will be collected. We expect this visit to take approximately 15 minutes. 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
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Visit 7 (Day 9 +/- 7):  Subjects will be asked to return to the clinic for Visit 7 for follow-up. Subjects will be 
asked to refrain from bathing, exercising, or showering starting at 12:01am of Visit 7.  Vital signs will be taken. 
Any adverse events since the last visit will be recorded.  A brief exam of the skin on subjects’ arms will be 
performed.  For subjects with AD, the severity of AD on each arm will be graded using the localized SCORAD, 
modified EASI, and global SCORAD and EASI grading.  Subjects will also be asked to grade their pruritus on a 
scale of 1-10, with 10 being the worst pruritus of their life.  Digital photographs of the lesional skin on both 
arms will be taken.  Next, one culture swab and one to two DNA swabs will be taken from the eczematous skin 
from each arm, and one culture swab and one to two DNA swabs will be taken from the non-lesional skin on the 
upper inner arm for a total of up to twelve (12) swabs. We expect this visit to take approximately 15 minutes. 
 
Visit 8 (Day 11 +/- 7):  Subjects will be asked to return to the clinic for Visit 8 for follow-up. Subjects will be 
asked to refrain from bathing, exercising, or showering starting at 12:01am of Visit 8.  Vital signs will be taken. 
Any adverse events since the last visit will be recorded.  A brief exam of the skin on subjects’ arms will be 
performed.  For subjects with AD, the severity of AD on each arm will be graded using the modified EASI, and 
global SCORAD and EASI grading.  Subjects will also be asked to grade their pruritus on a scale of 1-10, with 
10 being the worst pruritus of their life.  Digital photographs of the lesional skin on both arms will be taken.  
Next, one culture swab and one to two DNA swabs will be taken from the eczematous skin from each arm, and 
one culture swab and one to two DNA swabs will be taken from the non-lesional skin on the upper inner arm 
for a total of up to twelve (12) swabs. At the conclusion of this visit, subjects may resume prohibited 
medications, as needed, and may use their choice of soap and emollient. We expect this visit to take 
approximately 15 minutes. 
 
Visit 9, Phone 2 (Day 38  +/- 7 days) 
Subjects will be called approximately one month after their Visit 6.  During this phone call, subjects will be 
asked about any adverse events (including serious adverse events) since their last follow-up visit, as well as how 
their eczema is currently doing.  Although this phone contact will be the last scheduled contact for subjects with 
respect to this study, any subject with an ongoing adverse event (including serious adverse event) at the time of 
this phone contact will continue to be followed by study personnel until resolution of the event. 
8.4. Unscheduled Visits  
 
If disease activity increases or other concerns arise between regularly scheduled visits, participants should be 
instructed to contact study personnel and may be asked to return to the study site for an “unscheduled” visit.  
8.5. Visit Windows 
Study visits should take place within the time limits specified below: the designated visit windows (i.e. +/- n 
days) for each scheduled visit are also indicated on the Table of Events 
 

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Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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Investigator Name: Richard Gallo, MD, PhD 
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9.1 Skin swabs Samples 
9.1.1. Skin Swab collection 
The antecubital fossae bilaterally will be defined as the lesional swab location. The non-lesional swab location 
will be defined as the upper inner arm without AD skin. The designated site for swabbing will be marked with a 
pen and non-identifying digital photographs will be taken of the swabbing site for future visits.  The designated 
site  for swabbing will be measured for each subject, and the total area (cm x cm) recorded.   Using a skin swab, 
the lateral edge of the swab will be rubbed across the measured area in a cross-wise manner while rotating the 
swab handle between the thumb and forefinger.  Swabbing will be performed for at least 30 seconds.  Up to 6  
swabs may be obtained from each site. 
9.1.2. Staphylococcus aureus Screening assay 
Skin swabs will be collected from pre-measured areas of lesional and non-lesional skin per Section 9.1.1.  
Swabs will be resuspended in 1.5 mL tryptic soy broth (TSB) containing 15% (v/v) glycerol. Dilutions of 
culture medium will then  be used to inoculate on mannitol salt agar plates with egg yolk for selective growth of 
Staphylococcus species and differentiation of coagulase-positive and coagulase-negative strains.  Plates will be 
incubated for 24 hours in a 5% CO2 incubator at 37◦ C. The plates will be evaluated for the presence of S. 
aureus colonies, which appear yellow with a surrounding clear zone. 
9.1.3. 9.1.2. CFU Quantification of Live Staphylococcus aureus 
  
Skin swabs will be collected from pre-measured areas of lesional and non-lesional skin per Section 
9.1.1. Swabs will be suspended in 1.5 mL tryptic soy broth (TSB) containing 15% (v/v) glycerol and 
stored frozen at -80°C.  Live CFUs of coagulase positive S. aureus will be determined by inoculation on 
a bacterial culture plate containing mannitol salt agar with egg yolk for selective growth of 
Staphylococcus spp.  S. aureus will be conventionally distinguished from coagulase negative 
Staphylococcus according to mannitol metabolism and the egg yolk reaction, and coagulase positive and 
negative Staphylococcus will be quantified by manual counting of limiting dilutions of the stock 
solution.  
9.1.4. Quantification of Staphylococcus aureus, S. hominis, S. epidermidis, and total 
Staphylococcus DNA by q PCR 
To collect bacterial DNA, pre-measured areas similar to those used for bacterial culture  will be rubbed 
with a swab pre-moistened with Tris-EDTA buffer containing 0.1% TritonX-100 and 0.05% Tween-20 
(w/v).Bacterial-genomic DNA will be extracted for quantitative PCR  with the UltraClean Microbial 
DNA Isolation Kit (Mo Bio Laboratories:12224).  DNA will be eluted with 50uL DNAse free 
water.  0.5 µL of the elution will be used for quantitative real-time PCR (qPCR) using universal 
16SrRNA primers and species-specific primers. For quantification of S. aureus, S. epidermidis andS. 
hominis DNA, DNA extracted from an authentic number of each bacteria (ATCC35556, ATCC12228 
and ATCC27845, respectively) will be used for standard curve.  The standard curve will be used for 
determination of absolute number of bacteria (CFU/uL). This known amount of bacteria for S. aureus 
and CoNS will range from 1x 101 to 1x106 CFU/microliter.  The absolute number of bacteria will then 
be converted to relative CFU (rCFU)/cm2 based on pre-measured swab area. 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
Version Date: May 19, 2016 
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We have confirmed that each primer set does not cross-react to other species of bacteria.  In addition, we 
have confirmed specificity of primer sequence by BLAST search. 
  
For total 16SrRNA quantification, relative abundance will be calculated with the ΔΔCt method.  The 
following universal 16S primer set will be used to determine relative abundance of 16S rRNA gene 
according to the reference [J Clin Microbiol 43, 5332-5337 (2005)].  This universal primer set covers 
79% of Proteobacteria, 85% of Actinobacteria, 72% of Firmicutes, and 61% of Bacteroidetes.  Thus, the 
reactivity of this primer set differs depending on bacteria genus level.  Therefore, we cannot determine 
absolute abundance of 16S rRNA gene from bacterial mixture using a standard curve of bacterial DNA 
extracted from single species.  Accordingly, we will determine relative abundance of the total 16S rRNA 
gene. We will determine relative abundance of 16S rRNA gene by comparing it to baseline 
abundance.  Baseline will be set to 1 and relative abundance will be determined by the Delta-Delta-Ct 
method.   
  
 
 
10. Biospecimen Storage 
 
Instructions for sample preparation, handling, storage, and shipping are included in the MOOP. The Principal 
Investigator (PI) will be responsible for acknowledging and implementing all the regulations for classification, 
sample handling, packaging and labeling, permits or authorizations, and personnel training for shipment of 
biological and hazardous materials required for the conduct of this study. 
Isolated and speciated CoNS strains that have been chosen for use in the AMT cream will be stored at -80 
degrees C in 15% TSB-glycerol medium until ready to be used for formulating each subject’s AMT cream. 
Each subject will be assigned a unique numerical identifier.  All bacterial stocks will be stored at -80 degrees C 
in Eppendorf tubes. Each tube will be labeled with subject’s unique numerical identifier as well as a separate 
bacteria identifier linked to the bacteria stock in the tube.  The key explaining the contents of each of the 
bacteria identifiers to what they are will be stored in the laboratory notebook for the AMT study. 
Any bacteria stock that is unused after the completion of this protocol will be disposed of as per UCSD’s 
hazardous waste protocol.  

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Name of study: Establishing a Skin Microbiome Transplant 
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11. Criteria for Participant and Study Completion and Premature Study Termination 
11.1. Participant Completion 
This study will be defined as complete when all study procedures are completed, including the follow-up phone 
call to assess any adverse events.  
11.2. Participant Stopping Rules and Withdrawal Criteria 
Participants may be prematurely terminated from the study for the following reasons: 
1. The participant elects to withdraw consent from all future study activities, including follow-up. 
2. The participant is “lost to follow-up” (i.e., no further follow-up is possible because attempts to 
reestablish contact with the participant have failed).  
3. The participant dies.  
4. The Investigator no longer believes participation is in the best interest of the participant. 
11.3. Participant Replacement 
Participants who withdraw or are withdrawn will be replaced. 
11.4. Follow-up after Early Study Withdrawal 
If a participant is withdrawn from the study for any reason, the participant may be asked to complete a final 
visit by phone to assess any adverse events after AMT application. During this phone call, subjects will be 
asked about any adverse events (including serious adverse events) since their last visit, as well as how their 
eczema is currently doing.  Any subject with an ongoing adverse event (including serious adverse event) at the 
time of this phone contact will continue to be followed by study personnel until resolution of the event. Should 
the subject withdraw prior to completion of 7 days of cream application, subject will be asked to return to the 
clinic in order to return aliquots of unused and used cream as well as to assess any adverse events after AMT 
application. Should subjects experience an adverse event, subject will continued to be followed until resolution 
of the event as in above.  
11.5.  Study Stopping Rules 
The study may be prematurely terminated for the following reasons: 
Rules for stopping the entire study will include: 
 Any serious adverse event for which there is a reasonable possibility that the study product caused the 
serious adverse event. 
 The development of a serious adverse event for which attribution cannot be assessed as definitely 
unrelated to the study product in 10% or more of the study population. 
 The development of any severe (Grade 3) adverse events in 10% or more of the study population. 
If any of these criteria for halting the study are met, the study will not be resumed until the relevant information 
has been discussed with the FDA and the FDA concurs with resumption of the study. 
 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
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12. Safety Monitoring and Reporting 
12.2 Definitions 
An adverse event (AE) is any untoward medical occurrence in a subject during participation in the 
clinical study or with use of the experimental agent being studied. An adverse finding can include 
a sign, symptom, abnormal assessment (laboratory test value, vital signs, electrocardiogram 
finding, etc.), or any combination of these. 
 
A serious adverse event is defined by regulatory agencies as one that suggests a significant hazard or 
adverse event, regardless of the investigator or sponsor’s opinion on the relationship to investigational 
product.  This includes, but may not be limited to, any event that (at any dose): 
 
is fatal 
 
is life threatening (places the subject at immediate risk of death) 
 
requires in-patient hospitalization or prolongation of existing hospitalization 
 
results in persistent or significant disability/incapacity 
 
constitutes a congenital anomaly/birth defect 
 
is medically significant, in that it may jeopardize the subject and may require medical or 
surgical intervention to prevent one of the outcomes listed above.   
Any event that does not exactly meet this definition, but in the investigator’s opinion represents a 
significant hazard (eg, emergency room visit or outpatient surgery), can be assigned the “other 
significant hazard” in regards to reporting serious criteria. 
Additionally, important medical events that may not be immediately life threatening or result in death 
or hospitalization but that may jeopardize the subject or require intervention to prevent one of the 
outcomes listed above, or result in urgent investigation, may be considered serious.  Examples include 
allergic bronchospasm, convulsions, and blood dyscrasias. 
All subjects will receive verbal and written instructions with site contact information for whom to 
contact if they experience an AE associated with study procedures performed for this protocol or if the 
subject resumes their protocol-prohibited medication during the washout period.   
 
 
 
 
 EXPECTED RISKS 
 
Risks of applying microbial cream (Cetaphil® Moisturizing Lotion mixed with the subject’s endogenous 
bacteria): 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
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1. 
Skin infection 
 
The likelihood of subjects developing a skin infection is low given that we will be transplanting their non-
pathogenic autologous bacteria that have antimicrobial peptide-producing abilities.  Since we are 
transplanting the subject’s own endogenous bacteria back onto them, we know that these bacteria are 
present on the subject’s skin at baseline without causing an infection.  In addition, the bacteria strains to be 
transplanted onto the patient will be screened prior to putting it into the AMT cream to ensure that it is not 
a skin pathogen.  The known pathogen S. aureus that frequently colonizes AD skin will not even be 
considered as a potential bacteria to transplant because of its yellow color on mannitol-salt-agar plates, 
which isolated bacteria will be grown on.  Only red colonies (S. epidermidis) growing on mannitol-salt-
agar plates will be chosen and screened as potential bacteria to be transplanted onto the subject.  If 
anything, we expect the population of S. aureus to be reduced with the use of the AMT cream, since the 
AMT cream will contain only bacteria that are known to inhibit S. aureus.  Furthermore, we will be 
applying these bacteria topically and will exam all patients first to ensure that they do not have any cracks 
or excoriated skin on their arms before we apply it.  We will also avoid applying the microbial cream to 
the hand since the hand would be a likely source of spreading these bacteria onto other surfaces that other 
people may contact.  Should an infection occur (or should the subject be concerned about an infection), the 
subject should call Drs. Richard Gallo or Tissa Hata.  The subject would be treated according to the 
standard treatment after examination by Drs. Richard Gallo or Tissa Hata. The treatment will include 
topical and/or oral antibiotics (for infection) depending on the severity of the infection clinically, and 
Cetaphil® moisturizing lotion (for irritation and dryness), provided by Drs. Richard Gallo or Tissa Hata.  
The topical antibiotic of choice will be mupirocin.  The oral antibiotic of choice will depend on the 
individual subject’s medical history, including any possible contra-indications to certain medications due 
to allergies or other concomitant medication use, but doxycycline will be the first line oral antibiotic, 
followed by a cephalosporin or penicillinase-resistant penicillin. 
 
If the subject calls to report a possible infection during a time when the clinic is closed, the subject will be 
directed to the resident on call or closest urgent care facility for further management. 
 
2. Risk of medication withdrawal:  Discontinuation of a person’s AD medication prior to participating in 
this study and also the prohibition of using AD medications for periods of time during this study may 
result in worsening or temporary flaring of the patient’s AD.  
 
As listed in the inclusion/exclusion criteria (section 10), subjects with severe AD who may have difficulty 
tolerating periods without medication use will be excluded from participating.  Subjects enrolled in the 
study will be able to use over-the-counter moisturizers as needed.  Moisturizers play a role in helping to 
prevent and control AD flares, and therefore may be of use to subjects whose AD is worsening. The time 
during which subjects are unable to treat their AD during the study will be limited to the 7-days prior to 
each screening and treatment/follow-up visit for topical medications, and 28-days prior to each screening 
and treatment/follow-up visit for oral medications.  If for some reason severe worsening of AD occurs 
during a time in the study that subjects are not permitted to use their medication, the subject will be 
permitted to use their medication, however the upcoming visit must be rescheduled so that the required 7-
day and 28-day wash-out periods before each screening and treatment/follow-up visits are observed.  
Since the visit dates for the study are relatively flexible (the only real time-dependent visit is the Follow-
up Visit, which must occur approximately 24 hours after the Treatment Visit), rescheduling subject visits 
should not affect study results.  AD itself is a chronic condition characterized by periods of flaring and 
remission.  Any worsening of the subject’s AD would constitute a disease flare that should be correctable 
with the use of medications.  Flares of AD do not result in permanent deterioration of the subject’s 

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condition.  Finally, topical medication discontinuation must only be observed on the subject’s bilateral 
arms since this is the site being studied during the trial.  Subjects may treat the AD on all other body 
surfaces with topical medications throughout the study. 
 
3. Risk of loss of confidentiality:  Every reasonable effort will be made to keep records confidential.  
Research records will be stored in a secure building under lock and key of the study personnel.  Data 
stored in our computers are password protected.   Unless required by law, only the Study Doctors, the 
Study Team, and the UCSD Institutional Review Board will have access to confidential data which 
identifies subject by name. Because of the need to give these parties access to this information, absolute 
confidentiality cannot be guaranteed. 
12.2.1 Adverse Event (AE) 
 
Reporting Procedures for All Adverse Events  
The investigator is responsible for ensuring that all adverse events are properly recorded in the 
subjects’ records.  After signing of the informed consent form, all adverse events observed by the 
investigator or reported by the subject (whether or not attributed to investigational product), will be 
recorded in the case report form.   
 
The following attributes must be assigned by the investigator: description; dates of onset and 
resolution; severity; assessment of relatedness to investigational product, and action taken.  The 
investigator may need to provide follow-up information, medical records, and extracts from medical 
records. 
       
 UCSD IRB 
Unanticipated problems involving risk to participants or others (UPRs) are defined as any problem or 
event, which in the opinion of the Principal Investigator was: 1) unanticipated, 2) serious, AND 3) at 
least possibly related to the research procedures. If the event meets all 3 criteria for a UPR, it should be 
reported to the UCSD IRB within 10 working days. The following are events that meet the definition of 
UPR:   
1. Any serious event (including injuries, side effects, deaths or other problems) that in the opinion of the 
Principal Investigator was unanticipated, involved risk to subjects or others, and was at least possibly 
related to the research procedures.  
2. Any serious accidental or unintentional change to the IRB-approved protocol that alters the level of risk.  
3. Any deviation from the protocol taken without prior IRB review to eliminate apparent immediate hazard 
to a research subject. 
4. Any new information (e.g. publication, safety monitoring report, updated safety report), interim result or 
other finding that indicates an unexpected change to the risk/benefit ratio of the research.  
5. Any breach in confidentiality that may involve risk to the subject or others.  

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6. Any complaint of a subject that indicates an unanticipated risk or that cannot be resolved by the 
Principal Investigator.  
  
A brief summary of all UPRs as well as a brief summary of all adverse events will be submitted with the 
continuing review. 
 
Food and Drug Administration 
The Principal Investigator will notify the FDA directly in a written IND safety report of any adverse 
experience associated with the use of the drug that is both serious and unexpected or any finding from 
tests in laboratory animals that suggests a significant risk for human subjects including reports of 
mutagenicity, teratogenicity, or carcinogenicity.  Each notification will be made as soon as possible and 
in no event later than 15 calendar days after the initial receipt of the information. Each written 
notification will be submitted on FDA Form 3500A.  The Principal Investigator will also notify FDA by 
telephone or by facsimile transmission of any unexpected fatal or life-threatening experience associated 
with the use of the drug as soon as possible but in no event later than 7 calendar days after initial receipt 
of the information.  
 
On an annual basis as part of the update to the study IND, the Principal Investigator will submit to the 
FDA directly a narrative or tabular summary showing the following: 
• The most frequent and most serious adverse experiences by body system 
• A summary of all IND safety reports submitted during the past year 
• A list of subjects who died during participation in the investigation, with the cause of death for each 
subject 
• A list of subjects who dropped out during the course of the investigation in association with any 
adverse experience, whether or not thought to be drug related 
 
12.3 Grading and Attribution of Adverse Events 
12.3.1 Grading Criteria 
Clinical investigators will “grade” the severity of all AEs using the following scale; the results will be 
reported on the SAE form, if necessary. 
1 = MILD – aware of sign or symptom, but easily tolerated 
2 = MODERATE – discomfort enough to cause interference with usual activity 

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3 = SEVERE – incapacitating with inability to work or do usual activity 
4 = LIFE-THREATENING – refers to an event in which the patient was, in the view of the investigator, at 
risk of death at the time of the event.  It does not refer to an event which hypothetically might have caused 
death if it were more severe. 
5 = FATAL 
 
12.3.2 Attribution Definitions 
The relationship, or attribution, of an adverse event to the study therapy regimen or study procedure(s) 
will initially be determined by the site investigator and recorded on the appropriate AE/SAE CRF.  Final 
determination of attribution for safety reporting will be determined by NIAMS.  The relationship of an 
adverse event to study therapy regimen or procedures will be determined using the descriptors and 
definitions provided in Table 12.3.2.  
 
       
Table 12.3.2 Attribution of Adverse Events 
Code 
Descriptor 
Relationship (to primary investigational 
product and/or other concurrent mandated 
study therapy or study procedure) 
UNRELATED CATEGORY 
1 
Not related 
The adverse event is clearly not related:  there is 
insufficient evidence to suggest a causal 
relationship. 
 RELATED CATEGORIES 
2 
Possibly/ 
Probably 
The adverse event has a reasonable possibility to 
be related; there is evidence to suggest a causal 
relationship. 
3 
Definitely 
related 
The adverse event is clearly related. 
 
12.5 Reporting of Serious Adverse Events and Adverse Events 
Recording and Reporting Obligations for SAEs 
The investigator will provide the IRB appropriate information concerning any findings that suggest there 
has been an SAE related to the study.   
The investigator will notify the UCSD Human Research Protections Program of any unexpected, fatal or 
life-threatening experiences in writing no later than 10 working days of the event, according to standard 
IRB procedures.  All serious and medically significant adverse events considered related to the product 
by the investigator will be followed until resolved or considered stable.   

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All SAE’s will undergo expedited reporting to the NIAMs and the Independent Safety Officer through 
KAI within 48 hours of the investigator becoming aware of the event. 
 
SAEs that are unanticipated, serious, and possibly related to the study intervention will be reported to 
the IRB, FDA, and Independent Safety Officer in accordance with requirements. For the IND/IDE: 
• 7-day IND Safety Report (unexpected fatal or life-threatening AEs related to the intervention); a copy 
of the report sent to the FDA will be submitted to the Independent Safety Officer within 24 hours of 
FDA notification. 
• 15-day IND Safety Report (any other serious and unexpected AE related to the intervention); a 
copy of the report submitted to the FDA will be submitted to the Independent Safety Officer within 
24 hours of FDA notification. 
• Other AEs documented during the course of the trial will be included in the annual IND report. In the 
annual AE summary, the Independent Safety Officer Report will state that they have reviewed all AE 
reports. 
   12.6   
Pregnancy Reporting 
 
The investigator shall be informed immediately of any pregnancy in all study subjects or a partner of a 
study subject. A pregnant subject shall be instructed to stop taking study medication. The investigator 
shall counsel the subject and discuss the risks of continuing with the pregnancy and the possible effects 
on the fetus. Monitoring of the pregnant subject shall continue until the conclusion of the pregnancy.   
The investigator will report to NIAMS within 1 business day of notification of the event.  
12.8 Review of Safety Information 
12.8.1 Medical Monitor Review 
The Safety Officer shall receive bi-annual reports from the investigator compiling new and 
accumulating information on AEs, SAEs, and pregnancies recorded by the study site. 
In addition, the Medical Monitor (Tissa Hata M.D.) shall review and make decisions on the disposition 
of the SAE and pregnancy reports. 
12.8.2 Safety Officer Review  
 
12.8.2.1 Planned Safety Officer Reviews  
The Safety Officer shall review safety data at least yearly during planned Safety Officer (SO) 
Data Review Meetings. Data for the planned safety reviews will include, at a minimum, a listing 
of all reported AEs and SAEs.   

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The SO will be informed of an Expedited Safety Report in a timely manner  
12.8.2.2 Ad hoc Safety Reviews  
Triggers for ad hoc review: 
 Any serious adverse event for which there is a reasonable possibility that the study product 
caused the serious adverse event. 
 The development of a serious adverse event for which attribution cannot be assessed as 
definitely unrelated to the study product in 10% or more of the study population. 
 The development of any severe (Grade 3) adverse events in 10% or more of the study 
population. 
If any of these criteria for halting the study are met, the study will not be resumed until the relevant 
information has been discussed with the IRB, Safety Officer, NIAMS, and the FDA and these 
bodies concur with resumption of the study. 
After review of the data, the Safety Officer will make recommendations regarding study conduct 
and/or continuation. 
12.8.2.2.1 Temporary Suspension of enrollment and drug dosing for ad hoc Safety Review  
The following are events that could trigger a safety review, resulting in temporary suspension of 
enrollment and drug dosing  
 Any serious adverse event for which there is a reasonable possibility that the study product 
caused the serious adverse event. 
 The development of a serious adverse event for which attribution cannot be assessed as 
definitely unrelated to the study product in 10% or more of the study population. 
 The development of any severe (Grade 3) adverse events in 10% or more of the study 
population. 
A temporary halt in enrollment and drug dosing will be implemented if an ad hoc SO safety 
review is required.  No new participants will be consented during this time and subjects will be 
instructed to stop application of the cream until the IRB, FDA, and Safety Officer concur that 
study procedures may be resumed.  
13. Statistical Considerations and Analytical Plan 
13.1 
Overview  
We propose here a randomized, double-blind, placebo-controlled trial that will apply beneficial bacteria 
from the AD host onto lesional skin of AD patients, thus performing an autologous microbiome 
transplant. Up to 54 subjects with AD will be recruited.  
13.2 
Endpoints/Outcomes  

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13.2.1 Primary Endpoint(s)/Outcome(s) 
 The change in S. aureus abundance after 7 days of AMT cream by using skin swabs to determine the 
quantity of S. aureus using culture and qPCR.  
13.2.2 Secondary Endpoint(s)/Outcome(s) 
Secondary Lesional AD skin:  
1. The abundance of the AMT bacterial strains on lesional skin at baseline and after 1 week BID 
application of AMT.  
2. The quantity of transplanted bacteria on lesional skin after stopping AMT cream 1 day, 2 days, and 4 
days after last application.  
3. The change in abundance of Staph aureus on lesional skin from baseline to 1 day, 2 days, and 4 days 
after stopping AMT. 
4. Change in clinical assessments, including SCORAD, modified EASI, and EASI after 1 week BID 
application of AMT. 
5. Identify lesional skin microbiome by DNA sequencing from baseline and after 1 week BID application 
of AMT. 
6. Evaluate the lesional skin microbiome transcriptome by RNA-sequencing analysis from baseline and 
after 1 week BID application of AMT. 
Secondary Non-lesional skin:  
1. The abundance of Staph aureus at baseline and after 1 week BID application of AMT on non-lesional 
skin. 
2. The abundance of AMT bacterial strains on non-lesional skin at baseline and after 1 week BID 
application of AMT. 
3. The quantity of transplanted bacteria on non-lesional skin after stopping AMT cream 1 day, 2 days, and 
4 days after last application.  
4. The change in abundance of Staph aureus on non-lesional skin from baseline to 1 day, 2 days, and 4 
days after stopping AMT. 
5. Identify non-lesional skin microbiome by DNA sequencing from baseline and after 1 week BID 
application of AMT. 
6. Evaluate the non-lesional skin microbiome transcriptome by RNA-sequencing analysis from baseline 
and after 1 week BID application of AMT. 
 
 
13.3 
Measures to Minimize Bias  
Subjects will be randomized into a 2:1 allocation ratio of AMT cream to placebo. Both subjects and 
investigators making clinical assessments will be blinded.  
13.4 
Analysis Plan 
13.4.1 Analysis Populations. 
 
 
 
Screening Population:  The Screening Population will consist of all subjects who sign informed 
consent.  Subject disposition, demographics, and safety measures will be summarized for this 

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population.  Additionally, differences between screened and randomized subjects and between 
screened and not randomized subjects will be examined.     
Safety Population: All randomized subjects who provide any safety data will be included in the 
Safety Population.  A subject will not be required to complete all safety assessments to be 
included in this population.  Analyses of this population will be classified according to the 
treatment actually received. 
Efficacy Population: An intention-to-treat analysis will be conducted that includes all subjects 
who had at least one follow-up visit after intervention. A per-protocol analysis will also be 
conducted that includes all subjects who were compliant with the full intervention regimen with 
no major protocol violations. 
 
13.4.2 Primary Analysis of Primary Endpoint(s)/Outcome(s)   
  
Aim: The change in S. aureus abundance after 7 days of AMT cream by using skin swabs to determine 
the quantity of S. aureus using culture and qPCR. 
  
For each AD subject, the abundance of S. aureus will be calculated using CFU counts from baseline 
(day 28) to Day 35 after 7 days of AMT cream application or placebo application. Comparisons of S. aureus 
reduction will be made between AMT cream and placebo subjects using a two-tailed t-test for independent 
samples with type II error rate of 0.2 and type I error rate of 0.05. The sample size will include 18 subjects in 
the AMT cream arm and 9 subjects in the placebo arm. Comparisons of AMT cream versus placebo cream will 
be reported as means, standard deviations, medians, and minimum and maximum. An intention-to-treat analysis 
will be conducted that includes all subjects who had at least one follow-up visit after intervention. A per-
protocol analysis will also be conducted that includes all subjects who were compliant with the full intervention 
regimen with no major protocol violations. 
 
13.4.4 Analyses of Secondary and Other Endpoint(s)/Outcome(s) 
 
Secondary Endpoints, Lesional AD skin:  
1. The abundance of the AMT bacterial strains on lesional skin at baseline and after 1 week 
BID application of AMT.  
a. The abundance of live CoNS species on lesional skin will be compared from baseline 
(day 28) to Day 35 after 1 week BID application of AMT cream versus placebo. This 
will be calculated using a two-tailed t-test for independent samples with type II error 
rate of 0.2 and type I error rate of 0.05. 
2. The quantity of transplanted bacteria on lesional skin after stopping AMT cream 1 day, 2 
days, and 4 days after last application.  
a. Live antimicrobial Staph species will be measured on lesional skin at baseline (day 
28), Day 35 (after 7 days of cream application), Day 36, Day 37, and Day 39 in order 
to evaluate the abundance of the antimicrobial CoNS species that remains after 

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stopping cream application. In order to take into account the variance of the 
abundance of antimicrobial CoNS species found on each subject’s skin at baseline, we 
will perform CFU counts at each time point (Day 28, 35, 36, 37, and 38) and evaluate 
the percentage of antimicrobial species present at each time point. The percentage of 
antimicrobial species present at each time point will be compared between the AMT 
cream arm and placebo arm using a two-tailed t-test for independent samples with 
type II error rate of 0.2 and type I error rate of 0.05. 
3. The change in abundance of Staph aureus on lesional skin from baseline to 1 day, 2 days, 
and 4 days after stopping AMT. 
a. The abundance of live Staph aureus on lesional skin will be evaluated using CFU 
counting at baseline, 1 hour, and 4 hours after initial application of AMT or placebo 
cream, and after 4 and 7 days of cream application. At each time point, we will 
compare the AMT cream and placebo arms by using a two-tailed t-test for 
independent samples with a type I error rate of 0.05 and type II error rate of 0.2.  
4. Change in clinical assessments, including SCORAD, modified EASI, and EASI after 1 week 
BID application of AMT. 
a. A modified EASI, global SCORAD and EASI score will be assessed at baseline 1 
week BID application of AMT cream or vehicle cream. Using percentage of 
improvement of these scores, we will compare percent reduction of AD severity 
between AMT cream and placebo cream using a two-tailed t-test with type I error rate 
of 0.05.  
5. Identify lesional skin microbiome by DNA sequencing from baseline and after 1 week BID 
application of AMT. 
a. Using DNA sequencing, the microbiome of lesional skin will be identified at baseline 
and after 1 week BID application of AMT.  
6. Evaluate the lesional skin microbiome transcriptome by RNA-sequencing analysis from 
baseline and after 1 week BID application of AMT. 
a. Using RNA-sequencing analysis, the microbiome of lesional skin will be evaluated at 
baseline and after 1 week BID application of AMT.  
 
Secondary Endpoints, Non-lesional skin:  
 
1. The abundance of Staph aureus at baseline and after 1 week BID application of AMT on 
non-lesional skin. 
a. The abundance of Staph aureus on non-lesional skin will be compared from baseline 
(day 28) to Day 35 after 1 week BID application of AMT cream versus placebo. This 
will be calculated using a two-tailed t-test for independent samples with type II error 
rate of 0.2 and type I error rate of 0.05. 
2. The abundance of the AMT bacterial strains on non-lesional skin after 1 week BID 
application of AMT.  
a. The abundance of live CoNS species on non-lesional skin will be compared from 
baseline (day 28) to Day 35 after 1 week BID application of AMT cream versus 
placebo. This will be calculated using a two-tailed t-test for independent samples with 
type II error rate of 0.2 and type I error rate of 0.05. 
3. The quantity of transplanted bacteria on non-lesional skin after stopping AMT cream 1 day, 2 
days, and 4 days after last application.  

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a. Live antimicrobial Staph species will be measured on non-lesional skin at baseline 
(day 28), Day 35 (after 7 days of cream application), Day 36, Day 37, and Day 39 in 
order to evaluate the abundance of the antimicrobial CoNS species that remains after 
stopping cream application. In order to take into account the variance of the 
abundance of antimicrobial CoNS species found on each subject’s skin at baseline, we 
will perform CFU counts at each time point (Day 28, 35, 36, 37, and 38) and evaluate 
the percentage of antimicrobial species present at each time point. The percentage of 
antimicrobial species present at each time point will be compared between the AMT 
cream arm and placebo arm using a two-tailed t-test for independent samples with 
type II error rate of 0.2 and type I error rate of 0.05. 
4. The change in abundance of Staph aureus on non-lesional skin from baseline to 1 day, 2 
days, and 4 days after stopping AMT. 
a. The abundance of live Staph aureus on non- lesional skin will be evaluated using 
CFU counting at baseline, 1 hour, and 4 hours after initial application of AMT or 
placebo cream, and after 4 and 7 days of cream application. At each time point, we 
will compare the AMT cream and placebo arms by using a two-tailed t-test for 
independent samples with a type I error rate of 0.05 and type II error rate of 0.2.  
5. Identify lesional skin microbiome by DNA sequencing from baseline and after 1 week BID 
application of AMT. 
a. Using DNA sequencing, the microbiome of lesional skin will be identified at baseline 
and after 1 week BID application of AMT. 
6. Evaluate the lesional skin microbiome transcriptome by RNA-sequencing analysis from 
baseline and after 1 week BID application of AMT. 
a. Using RNA-sequencing analysis, the microbiome of lesional skin will be evaluated at 
baseline and after 1 week BID application of AMT.  
 
 
13.4.5 Analyses of Exploratory Endpoint(s)/Outcome(s) 
13.4.6 Descriptive Analyses  
 
 
Background and baseline demographic, physical examination, and subject disposition data will be tabulated and 
presented for all enrolled subjects by randomized treatment group.  Disposition will be tabulated according to 
the number and percentage of subjects who have:  signed informed consent, provided baseline/screening 
information, were randomized to receive AMT cream or placebo, and completed at least one follow-up visit 
after intervention. Continuous data (e.g., age, body weight, height) will be summarized descriptively by mean, 
standard deviation, median, and range.  Categorical data (e.g., gender, race, severity of disease) will be 
presented as enumerations and percentages. An intention-to-treat analysis will be conducted that includes all 
subjects who had at least one follow-up visit after intervention. A per-protocol analysis will also be conducted 
that includes all subjects who were compliant with the full intervention regimen with no major protocol 
violations.  
 

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13.5 
Interim Analyses  
Interim analyses will include distributions of AEs by subject population.  No efficacy analyses will be 
performed unless specifically requested by the SO.  Additional analyses may be requested by the 
NIAMS SO.   
13.7 
Sample Size Considerations  
 
Number of S. aureus colonies will be a continuous variable, and change in S. aureus abundance will be 
measured as the absolute difference in number of colony-forming units at specified time points.  
The sample size was calculated using G-Power Data Analysis software for a two-tailed t-test for independent 
samples, type II error rate of 0.2, and type I error rate of 0.05. The values used in the analysis were determined 
from our pilot study in which we quantified the reduction in S. aureus on the forearms of 5 atopic dermatitis 
patients twenty-four hours after treatment with a live microbiome transplant. Since the sample size was small in 
this study, we used the median and median average deviation as measures of central tendency. The median S. 
aureus present on the forearms of atopic dermatitis patients at baseline was 192.86 CFU/cm2 with deviation of 
191.86. We observed that a log-base 10 reduction by 1 of the number of bacteria present on the forearms to be a 
clinically significant reduction at 24 hours, with deviation of 21.42.  Using µ1 = 192.86, µ2 = 19.186, and 
ơ1=191.86, and ơ2=21.42, the total sample size necessary to see this difference would be n=24 for an unequal 
allocation of subjects 2:1 in the treatment to placebo groups.  
Assuming a drop-out rate of 20%, we propose a total n=27, with n= 18 in the treatment group and n= 9 in the 
placebo group. Assignment to treatment or placebo groups will be determined using simple randomization with 
an online random number generator.   
This sample size is reasonable and practical within the scope of this funding mechanism and recruitment 
capabilities. 
An intention-to-treat analysis will be conducted that includes all subjects who had at least one follow-up visit 
after intervention. A per-protocol analysis will also be conducted that includes all subjects who were compliant 
with the full intervention regimen with no major protocol violations.  
 
14. Identification and Access to Source Data 
14.1. Source Data 
Source documents and source data are considered to be the original documentation where subject information, 
visits consultations, examinations and other information are recorded.   Documentation of source data is 
necessary for the reconstruction, evaluation and validation of clinical findings, observations and other activities 
during a clinical trial.  
14.2. Access to Source Data 

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The site investigators and site staff will make all source data available to KAI, NIAMS,  as well as to relevant 
health authorities.  Authorized representatives as noted above are bound to maintain the strict confidentiality of 
medical and research information that may be linked to identified individuals. 
15. Protocol Deviations 
15.1. Protocol Deviation Definitions 
Protocol Deviation – The investigators and site staff will conduct the study in accordance to the protocol; no 
deviations from the protocol are permitted.  Any change, divergence, or departure from the study design or 
procedures constitutes a protocol deviation.  As a result of any deviation, corrective actions will be developed 
by the site and implemented promptly. 
 
Major Protocol Deviation (Protocol Violation) - A Protocol Violation is a deviation from the IRB approved 
protocol that may affect the subject's rights, safety, or well-being and/or the completeness, accuracy and 
reliability of the study data.  In addition, protocol violations include willful or knowing breaches of human 
subject protection regulations, or policies, any action that is inconsistent with the NIH Human Research 
Protection Program’s research, medical, and ethical principles, and a serious or continuing noncompliance with 
federal, state, local or institutional human subject protection regulations, policies, or procedures.  
 
Non-Major Protocol Deviation - A non-major protocol deviation is any change, divergence, or departure from 
the study design or procedures of a research protocol that does not have a major impact on the subject's rights, 
safety or well-being, or the completeness, accuracy and reliability of the study data. 
15.2. Reporting and Managing Protocol Deviations 
The study site principal investigator has the responsibility to identify, document and report protocol deviations 
as directed by the study Sponsor.  However, protocol deviations may also be identified during site monitoring 
visits or during other forms of study conduct review. Upon determination that a protocol deviation has occurred, 
the study staff will notify the site Principal Investigator and complete a Protocol Deviation form. Protocol 
deviations, as determined by the PI, will be reported to the appropriate review bodies including the IRB, SO, 
and FDA, who will review and approve the action plan that will be implemented as a result of the protocol 
deviation.  
16. Ethical Considerations and Compliance with Good Clinical Practice 
16.1. Statement of Compliance 
This clinical study will be conducted using good clinical practice (GCP), as delineated in Guidance for 
Industry: E6 Good Clinical Practice Consolidated Guidance, and according to the criteria specified in this 
study protocol.  Before study initiation, the protocol and the informed consent documents will be reviewed and 
approved by the IRB and FDA.  Any amendments to the protocol or to the consent materials will also be 
approved by the IRB and FDA before they are implemented. 
16.2. Informed Consent Process 

Investigator Name: Richard Gallo, MD, PhD 
Name of study: Establishing a Skin Microbiome Transplant 
Grant number: 1 R21 AR067547-01A1 
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The consent process will provide information about the study to a prospective participant and will allow 
adequate time for review and discussion prior to his/her decision.  The principal investigator or designee listed 
on the FDA 1572 will review the consent and answer questions.  The prospective participant will be told that 
being in the trial is voluntary and that he or she may withdraw from the study at any time, for any reason.  All 
participants (or their legally acceptable representative) will read, sign, and date a consent form before 
undergoing any study procedures.  Consent materials will be presented in participants’ primary language. A 
copy of the signed consent form will be given to the participant. 
The consent form will be revised when important new safety information is available, the protocol is amended, 
and/or new information becomes available that may affect participation in the study.  
16.3. Privacy and Confidentiality 
A participant’s privacy and confidentiality will be respected throughout the study.  Each participant will be 
assigned a unique identification number and these numbers rather than names will be used to collect, store, and 
report participant information.  Site personnel will not transmit documents containing personal health identifiers 
(PHI) to the study sponsor or their representatives. 
17. Publication Policy 
The NIAMS policy on the publication of study results will apply to this trial. 
18. References 
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Grant number: 1 R21 AR067547-01A1 
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Kim JE, et al. Selective antimicrobial action is provided by phenol-soluble modulins derived from 
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and Leung DY. Interleukin-10 downregulates anti-microbial peptide expression in atopic dermatitis. J 
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35. Howell MD, Gallo RL, Boguniewicz M, Jones JF, Wong C, Streib JE, and Leung DY. Cytokine milieu of 
atopic dermatitis skin subverts the innate immune response to vaccinia virus. Immunity. 
2006;24(3):341-8. 
36. Howell MD, Wollenberg A, Gallo RL, Flaig M, Streib JE, Wong C, Pavicic T, Boguniewicz M, and Leung 
DY. Cathelicidin deficiency predisposes to eczema herpeticum. J Allergy Clin Immunol. 
2006;117(4):836-41. 
37. LEUNG D, WOLLENBERG A, GALLO R, and al. e. Atopic dermatitis (AD) and eczema herpeticum (EH): 
Role of cathelicidins in herpes simplex virus (HSV) infection. JOURNAL OF ALLERGY AND CLINICAL 
IMMUNOLOGY. 2006;117(2):S187. 
38. Morioka Y, Yamasaki K, Leung D, and Gallo RL. Cathelicidin antimicrobial peptides inhibit 
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cytokine release and modulate chronic allergic dermatitis. J Immunol. 2008;181(6):3915-22. 
39. Gao PS, Rafaels NM, Hand T, Murray T, Boguniewicz M, Hata T, Schneider L, Hanifin JM, Gallo RL, Gao 
L, et al. Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema 
herpeticum. J Allergy Clin Immunol. 2009;124(3):507-13, 13 e1-7. 
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Hanifin JM, et al. The signal transducer and activator of transcription 6 gene (STAT6) increases the 

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propensity of patients with atopic dermatitis toward disseminated viral skin infections. J Allergy Clin 
Immunol. 2011. 
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