Experimental Study to Determine the Effects of Human Refluxate (Biliary, Gastric, Duodenogastric) on Macrophage Phenotype and Its Correlation With Different Forms of GERD: Motility Talent Group Study
Phase: NA
Conditions: Gastroesophageal Reflux Disease (GERD)
Sponsor: Ivashkin Vladimir Trofimovich

 
1.0   TITLE PAGE 
 
 
 
EXPERIMENTAL STUDY TO DETERMINE THE EFFECTS OF REFLUXATE 
(HUMAN: BILIARY, GASTRIC, DUODENOGASTRIC/MIXED) ON MACROPHAGE 
PHENOTYPE AND MACROPHAGE PLASTICITY AND ITS CORRELATION  WITH 
DIFFERENT  FORMS  OF ACID REFLUX DISEASE (NON-EROSIVE AND EROSIVE)  
AND COMPLICATIONS THEREOF  (BARRETT'S OESOPHAGUS): MOTILITY 
TALENT GROUP STUDY. 
Product Name: 
No  investigational  product  
Type of Study: 
Date: 
IIS (Human, Animal (in  vitro)) 
12.10.2014 
Version  
Principal Investigator 
(Sponsor):  
 
 
     
1.0 
Vladimir Ivashkin, MD, PhD, Professor 
Igor Malyshev,  MD, PhD, Professor 
 
 
 
 
 
 
 
 
Institution/Study Site:   
 
Sechenov First Moscow State Medical University, Russian Federation, Moscow, 8-2 
Trubetskaya st., Moscow 119991, Phone +7 (495) 622-96-43. 
 
Moscow State University of Medicine and Dentistry named after A.I. Evdokimov, Russian 
Federation, Moscow,  Building  1, 20, Delegatskaya st., Moscow, 127473, Phone +7 495 609 23 
66. 
 
   
 
This study   will be conducted in compliance with the protocol, Good Clinical Practice, 
Good Laboratory Practice, International  Guiding  Principles  for  Biomedical  Research  
Involving  Animal and  all other applicable regulatory requirements 

 
 
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1.0 
TITLE PAGE 
1 
2.0 
LIST OF ABBREVIATIONS 
3 
3.0 
INTRODUCTION 
4 
4.0 
RATIONALE 
7 
5.0 
PROGRAM OBJECTIVE(S) 
6 
5.1 
Study  Objectives 
7 
5.2 
Primary Endpoint 
7 
6.0 
INVESTIGATIONAL PLAN 
8 
6.1 
Selection of Program Population 
8 
6.1.1 
Inclusion Criteria 
8 
6.1.2 
Exclusion Criteria 
9 
6.1.3                Patient completion  and withdrawal                                                                       10 
6.2 
Number of Patients to be Enrolled 
10 
6.3 
Investigator Selection Criteria 
11 
6.4 
Program Duration 
10 
6.5 
Program Conduct 
11 
6.6 
Description of Activities 
                                                 14  
7.0 
SAFETY                                                                                                                25 
8.0                   ETHICS AND QUALITY                                                                                     25 
9.0 
STATICTICAL METHODS  
26 
10.0 
REFERENCES 
28 

 
 
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2.0  LIST OF ABBREVIATIONS 
APS 
American Physiological Society 
CD 
Cluster of differentiation 
GERD 
Gastroesophageal reflux disease 
GCP 
Good  clinical  practice 
GLP 
Good laboratory practice 
H2RA 
H2 receptor antagonists 
IBD 
Inflammatory bowel disease 
IL 
Interleukin 
LEC  
Local Ethics Committee 
M1 
Macrophage type 1 
M2 
Macrophage type 2 
NERD 
Non-erosive reflux disease 
рCRF 
Paper Case Report Form 
PPIs   
Proton-pump inhibitors 
Th1 
Type 1 T-helper 
Th2 
Type 2 T-helper 
TNFα 
Tumor necrosis factor alpha 
TNFβ 
Tumor necrosis factor beta 
 
 
 

 
 
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3.0 INTRODUCTION  
Gastroesophageal reflux disease (GERD) is one of the most common diseases and, according to 
recent epidemiological studies, clinical and endoscopic GERD symptoms can be detected in       
8-25% of the population depending on country, race and gender [1]. Although GERD rarely is a 
cause of death, it is associated with prominent morbidity and complications, such as esophageal 
ulceration, peptic stricture, Barrett`s esophagus and therefore leading cause of esophageal 
adenocarcinoma [2]. 
In the Russian Federation, the prevalence of GERD reaches 11-15% [2]. The incidence of GERD 
in the past 15 years has increased significantly and is currently 5 per 1000 population per year 
[1]. 
Despite improvements in diagnostics and treatment of GERD, there are still many unresolved 
issues. An example  of a diagnostic  challenge is identifying  patients with non-erosive reflux 
disease (NERD), which has been documented to occur in 50-60 % of  GERD patients. These 
patients periodically or continuously complain on heartburn, while they do not have endoscopic 
evidence of esophagitis. 
Another challenge concerns patients suffering from refractory GERD, who despite  treatment do 
not improve clinically and/or endoscopically, but on the contrary, progress to more extensive 
erosion, ulceration and esophageal complications such as peptic esophageal stricture, bleeding, 
and Barrett's esophagus which constitutes a precancerous disease [4]. 
In GERD, damage of  the esophageal mucosa may occur  as a result of  activity of different types 
of refluxate, i.e. gastric, biliary or duodenogastric/mixed. Along with acid and pepsin, duodenal 
contents with duodenogastric reflux may be harmful to the esophageal mucosa. Previous studies 
demonstrate that conjugated bile acids produce fewer harmful  effects than deconjugated bile 
acids in a more neutral pH environment [24]. Although proton pump inhibitors may weaken both 
acid and duodenogastric reflux by decreasing its volume, their prescription not always 
adequately addresses or eliminates the cause of the disease. 

 
 
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In recent years, the study of refractory GERD at the tissue and cellular level, which is based on 
the study of esophageal mucosal injury depending on the nature of   the  refluxate  (gastric, 
biliary or duodenogastric/mixed) has increasingly become an area of interest  particularly also in 
the light of investigating the immune response in patients with GERD. For instance, it appears 
that patients with high levels of interleukin-8 (IL-8) have a higher risk of recurrent GERD [5], 
and patients with high levels of Il-1b have a higher risk of torpid course of GERD [6]. 
Cytokines, important for cell signaling, are mainly produced by macrophages. Macrophages may 
change their phenotype under influence of different factors (known as macrophage plasticity). 
Macrophages produce not only inflammatory and anti-inflammatory cytokines, but  also  growth 
factors and reactive oxygen species (superoxide radical anions, singlet oxygen,  hydrogen 
peroxide), which define immune response of the whole body [7]. 
GERD is characterized by disorders in the immune response as  presented by misbalanced 
cellular (Th1) and humoral (Th2) parts of immune response, which might be determined by 
different phenotypes of macrophages – M1 (pro-inflammatory) or M2 (anti-inflammatory). 
Previous studies have shown that the activation of Th1-immune response leads to the erosive 
form of GERD, whereas activation of Th2- immune response may lead to the development of 
Barrett`s esophagus [8, 9]. 
 

 
 
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4.0 RATIONALE  
According to the published data, type of esophageal injury and its mucosal response greatly 
depends on the nature/type of refluxate (gastric, biliary or duodenogastric/mixed) and patient 
immune profiles [6].    It has been shown that Th-2-predominant cytokine response leads to 
Barrett`s esophagus. Also it was shown that acid suppression and alkaline duodenal reflux may 
increase the risk of Barrett`s esophagus [23].  
We hypothesize that the different types of refluxate may somehow differentiate macrophage 
phenotype and thereby the immune profile, determining the type of GERD disease and  its 
progression.           
This study is proposed to evaluate the role of motoric dysfunctions and burden of different kinds 
of refluxate on  immune cells and the subsequent course of the disease. This study also aims to 
establish a bridge between instrumental findings (pH-impedance test) and  clinical assessments 
(FSSG questionnaire), aiming to facilitate the  use of simple questionnaire techniques in the 
clinical setting in the future.  
 
This study will potentially also provide further insight and input into   future  uses and 
indications for  prokinetics in GERD and may aid  in  improving  the  treatment concepts of  
GERD and complications thereof (Barrett’s oesophagus). 
 
5.0 PROGRAM OBJECTIVE(S) 
5. 1. Study Objectives:  
 Animal (in  vitro) part of the study: 
 1. To  determine changes of macrophage phenotype and macrophage plasticity in mice, 
particularly C57/BL6 genetic strain after exposure to gastric refluxate (human origin) 
2. To determine changes of macrophage phenotype and macrophage plasticity in mice, 
particularly C57/BL6 genetic strain after exposure to biliary refluxate (human origin) 

 
 
7 
 
3. To determine changes of macrophage phenotype and macrophage plasticity in mice, 
particularly C57/BL6 genetic strain after exposure to duodenogastric/mixed refluxate (human 
origin). 
 
Clinical (patients) part of the study 
1. To investigate blood and  biopsy-origin  monocyte/macrophage phenotype in patients with 
different forms of acid reflux disease (non-erosive GERD (NERD), erosive GERD and  
complications  thereof: Barrett`s esophagus) 
2. To  investigate types of refluxate (gastric, biliary, duodenogastric/mixed) in patients with 
different forms of  acid reflux disease (non-erosive GERD (NERD), erosive GERD and 
complications thereof: Barrett`s esophagus) 
3. To  investigate the relationship between clinical assessments (FSSG questionnaire) and 
measurements obtained from esophageal pH-impedance monitoring and esophageal high-
resolution manometry. 
5.2. Primary Endpoint 
Animal (in  vitro) part of the study: 
To determine changes in macrophagal phenotype from baseline after exposure to three different 
forms of refluxate  (gastric, biliary duodenogastric/mixed) by means of assessing: 
a) Morphological characteristics (quantity of M1 (round forms) and M2 (fibroblasts-like) 
macrophages on 100 analyzing cells in 5 non-interfering sights). 
b) Macrophagal phagocytosis  activity (mean percentage of phagocytizing cells in all 
peritoneal macrophagal cells and mean number of bacteria ingested by macrophages) 
c) Secretory activity (ratio of proinflammatory M1 cytokines IL-1β, IL-8, IL-12p70, IFNγ, 
TNFα and TNFβ to  anti-inflammatory М2 cytokines IL-4, IL-5 и IL-10, as well as 
M1/M2 cytokines IL-2 и IL-6). 

 
 
8 
 
d) Receptor characteristics (ratio of superficial markers CD80 and CD 25 (M1-markers) and 
CD206 and CD163 (M2-markers). Changes in M1 and M2 ratios determined on the base 
of receptors will also be analyzed. 
 
Clinical (patients) part of the study: 
1. Characteristics of blood and  biopsy-origin    monocyte/macrophage phenotype (morphology, 
receptor characteristics) in patients with different types of acid reflux disease (NERD, erosive 
GERD) and complications thereof: Barrett's esophagus).  
2. Number of patients with gastric, biliary, duodenogastric/mixed  refluxate  within each of the 
different patient groups (NERD, erosive GERD and complications thereof: Barrett's esophagus). 
3. Mean FSSG scores (acid reflux related symptoms, dysmotility symptoms)   in patients with 
gastric, duodenogastric/mixed reflux related to the three different  patient subgroups (NERD, 
erosive GERD and complications thereof: Barrett's esophagus). 
 
 
6.0 
INVESTIGATIONAL PLAN 
6.1. Selection of Program Population 
Animal (in vitro) Part: Mice of genetic line C57/BL6 (90±15 mice) 
Clinical (patients) part:  90 adult patients with different types of acid reflux disease (30±5  
patients with non-erosive GERD, 30±5  patients with erosive GERD and 30±5 patients with 
complications thereof (Barrett’s esophagus)) 
 
6.1.1 Inclusion Criteria  
Animal (in  vitro) Part of study: 

 
 
9 
 
Mice of genetic lines C57/BL6 (black) with mean weight 24 g, mean age 10 weeks, males 
 
Clinical (patients) part of study: 
1. Signed  informed  consent 
2. Gender: Male or Female 
3. Age:  18-65 years of age 
3. Clinically and/or endoscopically confirmed diagnosis of GERD  
 
6.1.2. Exclusion Criteria  
Animal (in  vitro) Part: None 
Clinical: (patient) 
1. Treatment with PPIs and/or H2RA  1 week prior to study inclusion. 
2. Female patients who are pregnant, planning to become pregnant or  lactating 
3.  Any acute diseases or conditions, exacerbations of concomitant chronic diseases (including but 
not limited to inflammatory bowel disease (IBD), ulcer disease etc.)  at study start/inclusion   
and/or which  are   not   resolved  14 days prior  to study-enrolment.     
4. Participation in a clinical trial in the past 3 months 
5. Any condition which, in the opinion of investigator, makes the patient unsuitable for 
participation in the study 
6.1.3. Patient  completion   and withdrawal 
A patient who  performed  all visits will   be  considered   to  have  completed  the  study. 
A patient may voluntary discontinue  participation  in  the   study   at   any   time  and  for   any   
reason  without explanation. Nevertheless, the investigator  will make all  reasonable effort   to 
capture the reasons for  withdrawal.  

 
 
10 
 
The   investigator   may   also  at  his/her discretion discontinue   the  patient  from  participating    
in  this  study   at   any  time.(e.g. if  further  participation  in   the    study  is not considered to be  
in the interest of the patient : if major complications  of  the  procedures occur or  in  case of  
need  to   prescribe prokinetics 48 h or antacids 24h prior to esophageal  pH-impedance test).   
The   reasons   for   withdrawal   will be documented   in the primary data source data file 
(patient records) and CRF. 
 
6.2. Number of subjects to be enrolled 
Animal (in  vitro) Part of the study: 
М1 macrophages from mice of genetic line C57/BL6 (90±15 mice) – 90±15 samples 
i.e. number of laboratory sockets:  180±30 (90±15 samples in double) 
 
Clinical (patients) Part of the study: 
30±5 patients with non-erosive GERD (NERD, as confirmed  by endoscopy) 
30±5  patients with erosive GERD 
30±5  patients with Barrett`s oesophagus 
6.3. Investigator Selection Criteria: N/A 
6.4. Program Duration 
6.4.1. Сlinical  part (patients)  of  the  study: 
Program duration for each patient will be approximately 7( ±3 ) days. Duration of recruitment  is 
planned to be 14 months, 4  months  is  planned   for  Data Analysis, 6  months – for  study  
report/ publication preparation.   

 
 
11 
 
6.4.2. Animal (in vitro) part  of the study 
Duration of in vitro part of the study for each sample will be defined by: 
- period of macrophage culture – 48 hours,  
- macrophage phenotype assessment – 24 hours. 
The total duration of the in vitro study to obtain  one sample will be 36 hours. 
6.5. 
Program Conduct 
This  is a   prospective, experimental   study   consisting   of  the  two parts:  a clinical (patients)  
part and  an  animal (in  vitro) part. 
The clinical (patients)  part of the study will be conducted at  the   Propaedeutic Clinic of   
Internal medicine, Gastroenterology, Hepatology  of   First MGMU n.a.. IM Sechenov.    
The animal part of the study (in  vitro)  will be conducted at the  Moscow State University of 
Medicine and Dentistry.  
The in vitro part of the study will be conducted in М1 macrophages from mice of genetic line 
C57/BL6 (90±15 mice). To mirror the design of clinical part of the study and to ensure 
compliance with the enrolled patients’ number macrophages from 30±5 mice will be exposed to 
acid refluctate, macrophages from another 30±5 mice – to biliary refluctate and from the other 
30±5 mice – to mixed refluxate. 
 
 
 
 
 
 

 
 
12 
 
Figure 1. Study design 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 1. List of the  study  procedures (clinical part).  
Patients (n=90) with 
clinically and/or 
endoscopically confirmed 
GERD 
Upper gastrointestinal 
endoscopy with  the 
collection of refluxate  
M1 peritoneal macrophages 
from mice of genetic line 
C57/BL6 
Gastric 
refluxate 
Determine of changes from 
baselines of macropphagal 
phenotype after exposure to 
gastric refluxate by means 
of morphological and 
receptor 
characteristics, secretory  
and phagocytosis activity   
Determine of changes from 
baseline of macropphagal 
phenotype after exposure to 
duodenogastric/mixed 
refluxate by means of 
morphological and receptor 
characteristics, secretory  and 
phagocytosis activity   
Determine of changes from 
baseline of macropphagal 
phenotype after exposure to 
biliary refluxate by means 
of morphological and 
receptor 
characteristics, secretory  
and phagocytosis activity   
Biliary 
refluxate 
Duodenogastric/mixed 
refluxate 

 
 
13 
 
 
* Any  safety  issues  related   to   the  invasive study   procedures 
 
6.6.  Description of Activities 
Visit 1 (Baseline, Day 0) 
 
             
 
Visit 1 
 
 
Visit 2 
Visit 3 
Visit 4 
 
 
Visit 5  
Timelines and Allowance 
Window 
Day 1 
Baseline 
 
Day 2 
(±1Day) 
Day 3 
(±2 Days) 
Day 4 
(±2 Days) 
 
Day 5                 
Signed informed  consent 
X 
 
 
 
 
Assignment of patient 
identification number   
X 
 
 
 
 
 
 
Inclusion and exclusion 
criteria assessment 
X 
 
 
 
 
Allocation   to   the  
subgroups of GERD 
 
 
X 
 
 
Demographics (date  of  
birth, gender, race) 
X 
 
 
 
 
Medical history  
X 
 
 
 
 
Physical examination  
X 
X 
X 
X 
X 
FSSG assessment 
X 
 
 
 
 
Blood sampling 
(macrophages) 
 
X 
 
 
 
Hematology (RW, HIV, 
HbsAg, HCV) 
 
X 
 
 
 
Upper gastrointestinal 
endoscopy with  biopsy and 
collection of refluxate   
 
 
X 
 
 
24-h esophageal pH-
impedance test with  
esophageal high-resolution 
manometry 
 
 
 
X 
 
Safety assessments* 
X 
X 
X 
X 
X 

 
 
14 
 
The  procedures  of visit: 
• 
 Signed informed  consent 
Patients   considered eligible   will   be   informed   about   the  general  aspects   and  procedures  
of  the   study  and   have to sign      informed   consent   prior to any assessment or procedure is 
conducted.  
• 
Assignment of patient identification number  
Each patient will be assigned a two-digit  patient identification  number (ascending from 01 to 
90). 
• 
Inclusion / exclusion criteria assessment    
The  following  criteria  should  be  checked  prior to study enrollment:  
Inclusion criteria: 
1. Signed informed  consent 
2. Gender: Male or female 
3. Age:  18-65 years of age 
3. Clinically and/or endoscopically confirmed diagnosis of GERD 
 
Exclusion criteria  
1. Current treatment or treatment with PPIs and/or  H2RA 1 week prior inclusion  
2. Female patients who are pregnant, planning to become pregnant or  lactating 
3.  Any acute diseases or conditions, exacerbations of concomitant chronic diseases (including but 
not limited to inflammatory bowel disease (IBD), ulcer disease etc.)  at study-start/ inclusion  
and/or which  are   not   resolved 14 days  prior to  enrolment   in   the   study.    
4. Participation in a clinical trial in the past 3 months 

 
 
15 
 
5. Any condition which, in the opinion of investigator, makes the patient unsuitable for 
participation in the study 
• 
Demographics  
Includes information about date of birth, gender, race. 
• 
Medical history  
Medical history includes full medical  information regarding   GERD (duration of symptoms, 
results of previous examinations (upper gastrointestinal endoscopy, 24-h oesophageal pH-
impedance or pH test, esophageal high-resolution manometry ), previous and current treatment, 
comorbidities  and  concomitant  medication, family history of the disease. Historic reports of 
undesirable events associated with past medications will be considered medical history.  
•     Physical examination  
Includes the measurement of height, weight, blood pressure, pulse. 
•     FSSG assessment 
FSSG (Frequency Scale for the Symptoms of GERD) – questionnaire with 12 questions related 
to acid reflux and dysmotility symptoms (See Appendix A). Questions 2,3,5, 8 and 11 are related 
to `dysmotility`, while other questions consider acid reflux symptoms. FSSG allows investigators 
to analyze different symptoms experienced by GERD patients [10]. Sum of both parts of the 
questionnaire will be analyzed for each patient. Permission to use the questionnaire for this  
study was obtained from   its  author/developer, professor Motoyasu Kusano. 
 
• Safety Assessments 

 
 
16 
 
Any  AEs  related   to   the  invasive study   procedures, will   be   collected.                                                           
Only undesirable effects that are deemed at least possibly related to an Abbott medication by the 
investigator will be forwarded to Abbott affiliate PV. 
 
Visit 2   
•    Physical examination 
 Includes the measurement of blood pressure, pulse 
•   Hematology (RW, HIV, Hbs-Ag, HCV) 
Blood tests for  RW, HIV, Hbs-Ag, HCV are mandatory and considered  standard of medical 
care before  any interventional procedures are performed (such as endoscopy and pH-
impedancemetry).  
•  Blood sampling (monocytes/macrophages) 
The protocol of monocytes/macrophages isolation from venous blood 
1. Blood will be collected in previously marked Vacuette system test tube (10-12 ml) 
2. Blood in the test tube will be mixed with heparin (1 ml heparin – 10 ml blood) 
3. Transportation of the marked test tubes in the laboratory of Cellular Biotechnology of 
Moscow State University of Medicine and Dentistry. The samples will be stored at  
temperature +4±2оС until transportation and will be transported in a mobile refrigerator 
(+4±2оС) to the laboratory of Cellular Biotechnology of Moscow State University of 
Medicine and Dentistry. Laboratory address: 105275, Moscow, Boris Zhigulenkova 
street, 23, building 1 
4.  Blood sample is stirred and placed into centrifuge. 

 
 
17 
 
5. Blood sample is centrifuged for 15 minutes at 3000 rpm. 
6. After centrifugation the supernatant is separated from blood clot, in blood clot leukocytes 
are separated from erythrocytes, and leukocytes are placed in a pure plastic test tube. 
7. Lysis buffer is prepared: 100 ml of distilled water per 0.825 g NH4Cl, 0.0037 g EDTA. 
8. The leukocyte mass is mixed with 10 ml of lysis-buffer and mixed in vortex for 5 minutes 
(40 rpm). 
9. The test tube is centrifuged for 4 min – 1000 rpm.  
10. The supernatant is separated and drawn away. 
11. We place 5 ml of  growth medium RPMI1640 to the cell pellet and the tube is centrifuged 
for 4 min – 1000 rpm.  
12. The supernatant is separated and the cell pellet is resuspended in 1 ml RPMI1640.  
13. The cells are counted in a cell counter 
• Safety Assessments 
Any  AEs  related   to   the  invasive study   procedures, will   be   collected.                                                           
Only undesirable effects that are deemed at least possibly related to an Abbott medication by the 
investigator will be forwarded to Abbott affiliate PV. 
Visit 3  
•    Physical examination 
 Includes the measurement of blood  pressure, pulse 
 •    Upper gastrointestinal endoscopy with  biopsy  and collection of different types of 
refluxate. All   patients  will undergo an   upper gastrointestinal endoscopy with biopsy and 

 
 
18 
 
collection of different types of refluxate. The procedure   should   be   performed  on an empty 
stomach standard technique of  anesthesia;10% lidocaine solution).  The examination includes 
analysis of esophageal  mucosa, expression of inflammatory changes, location, size, number of 
mucosal  defects (erosions, ulcers, strictures, hemorrhages), as  well as  the appearance  of  the 
gastric and duodenal mucosa.  .  The modified Savary-Miller classification of esophagitis  will   
be  used  to  characterize   changes in  the  esophageal  mucosa. 
Table 2.  The  modified Savary-Miller classification of esophagitis  
I 
single erosive or exudative lesion, oval or linear, taking only one longitudinal fold 
II 
noncircular multiple erosions or exudative lesions taking more than one longitudinal fold, 
with or without confluence 
III circular erosive or exudative lesion 
IV chronic lesions: ulcer(s), stricture(s), erosive or exudative lesion or short oesophagus, 
isolated or associated with lesions of grades I, II, or III 
V 
islands, finger-like forms or circumferential distribution of Barrett’s epithelium isolated or 
associated with lesions of grades I to IV 
  
For morphological research  and  monocyte/macrophage phenotype assessment   biopsy 
sampling of distal esophagus will be  performed according to the  scheme: 4 fragments located at 
12, 3, 6, 9 hours, 2 cm above the Z-line, as well as from individual foci   of  altered mucosa. 
The   refluxate samples  required for the animal (in vitro) part of the study,  will   be    placed   
into  pure plastic test tubes and   be  stored   at  temperature +2-4 C until  transportation   to   the 
Cellular Biotechnology of Moscow State University of Medicine and Dentistry. Transportation  
should  be done  at  the   temperature     +2-4 C and be done within  two   hours after obtaining   
the  refluxate. 

 
 
19 
 
 Allocation to  the  subgroups of GERD  
Diagnosis of GERD (non-erosive and erosive)  is   made   based on   clinical symptoms 
(complaints   of heartburn, regurgitation, belching, (non-cardiac) chest pain) and/or  the   results  
of an  upper gastrointestinal endoscopy.  Endoscopy should not have been done more than a 
month before inclusion.  Diagnosis of Barrett’s esophagus has to be endoscopically confirmed 
(incl. biopsy) and should not be older than 6 months prior to study enrolment.  
Patients included into the study with a GERD diagnosis based on symptoms only and/or with an 
endoscopy performed more than one month ago prior to inclusion will be allocated   to   the   
subgroups    on    the   result   of   endoscopy  conducted  at Visit 3. 
• Safety Assessments 
Information regarding  complications  of  the  procedure  and patient complaints are to be 
collected and registered. After   the    procedure   each  patient will  be  placed under  medical  
observation  for  at least   for  two hours. Any  AEs  related   to   the invasive study   procedures, 
will   be   collected.                                                                                                                         
Only undesirable effects that are deemed at least possibly related to an Abbott medication by the 
investigator will be forwarded to Abbott affiliate PV 
Visit 4  
•  Physical examination 
 Includes the measurement of blood pressure, pulse. 
• 24-h esophageal pH-impedance test with esophageal high-resolution manometry  
To  investigate number of reflux episodes and determine the type of refluxate (gastric, biliary, 
duodenogastric/mixed) in patients with different forms of  acid reflux disease and complications 
thereof (Barrett`s esophagus)   pH-impedance test with esophageal high-resolution manometry 
(HRM) will  be done. 

 
 
20 
 
Calibration of device by means of two standard buffer solutions at temperature 37 C is necessary 
before the 24-hr esophageal pH-impedance test is performed.    
Procedure of  pH impedancemetry includes intranasal  introduction of a probe into  oesophagus  
and   connection of the  probe with the recording device and 24-hour wearing of  the  probe. 
Installation of the probe is carried out on an empty stomach.  
High-resolution manometry (HRM) will be done with a 22-channel water-perfused catheter and 
Solar GI system (Medical Measurements Systems, Enschede, the Netherlands). 
In GERD patients will be analyzed following HRM parameters: the distal contractile integral 
(DCI) and the lower esophageal sphincter resting pressure (LES RP). 
24-h pH-impedance monitoring will be done in both distal oesophagus and in the stomach 
according to standard of practice.  Esophageal pH sensor will be positioned 5 cm above the 
manometrically located border of the LES. Measurement with a second pH sensor in the 
stomach, 10cm below the LES in order to monitor intragastric acidity will be done as a standard 
routine procedure.    
The probe is removed after   24  hours and the results from the recording unit are transferred to 
the computer database for further analysis. 
Standard measuring parameters will be collected: percent of time when pH is less than 4, total 
number of reflux episodes, number of acid and alkaline reflux episodes,  duration of longest 
reflux episode.  
• Safety Assessments 
Information regarding  complications  of  the  procedure  and patient complaints are  collected 
and registered. After   the    procedure    each  patient  is placed   under  medical  observation  for  
at least  two hours. Any  AEs  related   to   the invasive study   procedures, will   be   collected.                             
Only undesirable effects that are deemed at least possibly related to an Abbott medication by the 
investigator will be forwarded to Abbott affiliate PV 
 

 
 
21 
 
 
Visit 5   
 Removing  of  the    device   for   24-h esophageal pH-impedance test.   
The results from the recording unit are transferred to the computer database for further analysis. 
 Physical examination 
 Includes the measurement of blood pressure, pulse. 
 Safety Assessments 
Information regarding  complications  of  the  procedure  and patient complaints are  collected 
and registered.  
Any  AEs  related   to   the invasive study   procedures, will   be   collected.                              
Only undesirable effects that are deemed at least possibly related to an Abbott medication by the 
investigator will be forwarded to Abbott affiliate PV. 
 
 
Conduct of the Animal (in-vitro) part of  the  study. 
The animal part of the study will be conducted in accordance with all applicable regulatory 
requirements: International  Guiding  Principles  for  Biomedical  Research  Involving  Animal, 
APS's Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training, 
Good Laboratory Practice. 
C57/BL6 mice selected for the study would be of an appropriate genetic strain and quality, and 
the minimum number  of animals required to obtain scientifically valid results will be defined 
according to the number of patients. Mice will be housed in the accredited vivarium of Moscow 
State University of Medicine and Dentistry to ensure that the general health of the animals is 
safeguarded and that undue stress is avoided. Special attention will be given to the space 

 
 
22 
 
allocation for each animal, and adequate standards of hygiene should be maintained as well as 
protection against predators, vermin, and other pests. Facilities for quarantine and isolation will 
be provided. Entry should normally be restricted to authorized persons. Environmental needs 
such as temperature, humidity, ventilation, lighting, and social interaction will be consistent with 
the needs of the species concerned. Noise and odor levels will be minimal. Proper facilities will 
be provided for the disposal of animals and animal waste. Animals will receive a supply of 
foodstuffs appropriate to their requirements and of a quality and quantity adequate to preserve 
their health, and they will have free access to potable water, unless the object of the experiment 
is to study the effects of variations of these nutritional requirements. Veterinary care, including a 
programme of health surveillance and disease prevention, will be available. Sick or injured 
animals should, according to circumstances, either receive appropriate veterinary care or be 
painlessly killed. 
All mice will be infused  2 ml of 4% thioglycolates 4 days before the experiment starts. All mice 
will receive etherization before the experiment. Peritoneal macrophages will be isolated from 
peritoneal fluid of C57/BL6 mice. Peritoneal lavage fluid will be centrifuged for 4 minutes - 
1000 rpm, then supernatant and cell pellet will be divided. Peritoneal macrophages will be 
cultured in RPMI1640 culture medium with 10% fetal bovine serum (FBS) and 100 U/ml 
penicillin and 100 µg /ml streptomycin on 48-well plate by 0.5x106 macrophages/well. 
In vitro experiments on animal  biological material (according to GLP  and  International  
Guiding  Principles  for  Biomedical  Research  Involving  Animal). 
Isolation and cultivation of macrophages. Macrophages will be isolated from biological 
material during  centrifugation at 1000 rpm within 4 minutes. The supernatant will be rejected, 
and the packed cells will be resuspended in RPMI-1640 nutritional medium. Then the 
concentration cells in RPMI 1640 will be make up to 1x106/ml. [11]. After the procedure of 
macrophages isolation  the degree of macrophages survival should be no less than 95% (a 
standard technique with trypan blue). Cell suspension will be distributed in the 48-well plate 
expected 0.5х106 cells per well containing 0.5 ml of RPMI 1640 medium. Macrophage 
cultivation will be carried out at 37оС and 5% CO2. 

 
 
23 
 
Determination of macrophage phenotype. At least there are three types of markers for 
definition of macrophages phenotype: 1) morphological marker, such as form of the cells, 2) 
receptor markers, such as CD25, CD80, CD163 and CD206 expression [12], and 3) functional 
markers, such as cytokine production or phagocytosis ability of cells [13]. Therefore we can 
define at least three types of macrophage phenotypes -  morphological, receptor and functional. 
We will execute an assessment of all three types of indicators. 
The morphological macrophage phenotype  was defined with the use of an ordinary light 
microscope with magnification power 15х40. Quantity of rounded-form macrophages, typical for 
M1 phenotype, and fibroblast-like cells, typical for M2 phenotype [13-18] was calculated among 
100 cells in 5 fields of vision in a well [19]. Rounded cells had an equal ratio of length to width 
or less than 2:1. If the ratio of length to width was more than 2:1, the form of a cell was regarded 
as fibroblast-like [20]. The morphological phenotype of macrophage population will be 
estimated as a ratio of absolute count of rounded-form macrophages to fibroblast-like ones. 
The receptor macrophage phenotype was determined by percentage of expression of superficial 
and cellular receptors - CD80 for M1 phenotype and CD206 for M2 phenotype [12, 13], in the 
isolated macrophages during flow cytometry  by means of CD80 and CD206 monoclonal 
antibodies [Beckman Coulter, USA; BD Pharmingen, USA]. Analysis was performed according 
to the producers’ instructions. The receptor phenotype of macrophage population will be 
estimated as a ratio of CD markers expression typical for M1 phenotype and CD markers of M2 
phenotype [18, 21]. 
The functional macrophage phenotype was estimated by cytokine (pro-inflammatory, such as IL-
12 (M1 markers) and anti-inflammatory, such as IL-10 (M2 markers)) production  [22]. Cytokine 
concentrations will be defined by flow cytometry [Beckman Coulter FC500, USA] with 
multiplex definition kits. The functional phenotype of macrophage population will be estimated 
as a ratio of functional markers of M1 and M2 phenotypes. 
 
Determination of macrophage phenotype after exposure to human acid refluctate, human 
biliary refluctate and mixed human refluctate will be performed by the above mentioned methods 
(the part of “Determination of macrophage phenotype”) and the changes vs initial results will be 
registered. 

 
 
24 
 
 
In vitro experiments on human biological material (according to GLP, GCP): 
 
Isolation and cultivation of macrophages. Macrophages will be isolated from biological 
material (blood) or derived from blood monocytes. Macrophages will be resuspended in RPMI-
1640 nutritional medium. Then the concentration of cells in RPMI 1640 will be adjusted to 
reflect a concentration of   1x106/ml. [11]. After macrophage isolation the degree of macrophage 
survival should not be  less than 95% (standard technique with trypan blue). The cell suspension 
will be distributed in a 48-well plate, with each well containing an expected 0.5х106 cells per 
well containing 0.5 ml of RPMI 1640 medium. Macrophage cultivation will be carried out at 
37оС and 5% CO2. 
 
Determination of macrophage phenotype will be performed using the   same  methods   as for  
animal  biological materials.    
Macrophage plasticity will be defined by standard criteria similar to  macrophage phenotype 
detection.  We will use the definition of morphological phenotype (ordinary light microscope 
with magnification power 15х40), receptor phenotype (percentage of expression of superficial 
and cellular receptors - CD80 for M1 phenotype and CD206 for M2 phenotype [12, 13], in the 
isolated macrophages during flow cytometry  by means of CD80 and CD206 monoclonal 
antibodies [Beckman Coulter, USA; BD Pharmingen, USA].)  and functional phenotype (NO 
and cytokine (pro-inflammatory, such as IL-12 (M1 markers) and anti-inflammatory, such as IL-
10 (M2 markers)) production  [22]. After determining the macrophage phenotype (see 
«Determination of macrophage phenotype»), the phenotypic plasticity (PhP) will be calculated. 
PhP of M1 will be calculated as percent change in the M1/M2 ratio under the action of M1 
reprogramming factors  and PhP of M2 will be calculated as percent change in the M2/M1 ratio 
under the action of M2 reprogramming factors. The phenotype marker ratio in standard culture 
conditions (10% FBS) will be taken as 100%. 
 
 

 
 
25 
 
7.0 
  SAFETY 
There  will  be  no investigational  product  in  the  study.  
It   is  planned  to  assign  the   standard  treatment    for  GERD   and   complications thereof  
(Barrett's oesophagus) on  the  measurement results of 24-h esophageal pH-impedance test  - on  
the   next  day  after visit  #5.   
However   some  medication    may   be   prescribed  before  the  study  completion in case  of  
need  and  in  the   interest  of  patient.    In case any Abbott product will be part of the patient’s 
treatment regimeany safety issue or safety signal, which would negatively impact the benefit-risk 
ratio of the concerned Abbott product and that is detected during conduct of the study is to be 
reported promptly to the Affiliate safety representative. 
 
8.0  ETHICS AND QUALITY 
The   study  will   be   conducted   in accordance  with  protocol   and  all   applicable   regulatory   
requirements: ICH Guideline   for  Good Clinical Practice (GCP), guiding  principles  of  the 
Declaration of Helsinki,  guiding  principles  of  Good Laboratory  Practice (GLP), International  
Guiding  Principles  for  Biomedical  Research  Involving  Animal and all applicable local 
regulations. 
Patient’s written authorization for use and/or disclose personal and/or health data must be 
obtained prior to enrolling each patient in the program and patients not willing to provide such 
written authorization will not be included in the program. However, all reasonable efforts will be 
made in order to avoid subject identifying information (such as name, address, etc.). 
The protocol and relevant program documents will be submitted for review and approval of 
Regulatory Authorities (if applicable) and  Local Ethics Committee(s) (LECs) and   the  study  
will start only after obtaining written approval from the  Regulatory Authorities (if applicable)  
and  LECs of   both Institutions. 

 
 
26 
 
All data will be captured and handled in such a way so as to not reveal identity of individual 
patients and hence patient confidentiality will be maintained at all times. 
The physician/investigator will be responsible for ensuring that a quality control and quality 
assurance system is in place to ensure that the program is conducted and data generated, 
documented, and reported in compliance with the protocol, accepted standards of Good Clinical 
Practice, and any applicable local laws and regulations. The  investigators  shall   be  solely   
responsible for  regulatory and legal compliance. 
The animal part of the study will be conducted in accordance with all applicable regulatory 
requirements: International  Guiding  Principles  for  Biomedical  Research  Involving  Animal, 
APS's Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training, 
GLP (as  described  above). 
9.0  STATISTICAL METHODS  
This is an experimental, interventional, non-randomized study.  The primary purpose of the this 
study is exploratory in its general sense and no hypothesis is being tested, therefore no sample 
size calculation was performed.  
Planned  enrollment  in  this   study constitutes  90 patients  in  clinical  part  and  90 subjects 
(mice) in  animal (in vitro)  part. 
Statistical analysis will be performed using the STATISTICA 8.0 software for Windows 
(StatSoft Inc, the USA).  
Normality of  data distribution  will  be tested by using either the Shapiro-Wilk or Kolmogorov-
Smirnov test.  
 In case  of normal (Gaussian) distribution  of  data  significance of differences will be 
determined by Student’s t-test. In cases with non-normal distribution we will use non - 
parametrical U-test according to the method of Mann-Whitney for independent samples.  
Tests resulting in p-values less than or equal to 0.05 will be reported as "statistically significant». 

 
 
27 
 
Continuous data will be summarized using descriptive statistics, and the following parameters 
will be reported:  number of observations (n), mean and standard deviation (SD); minimum 
(Min), and maximum (Max); median. 95 % СI  will  be calculated. 
 Categorical data will be presented using frequency (n) and percentage (%).   
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
28 
 
10.0  REFERENCES 
 
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3. Philip O. Katz, Lauren B. Gerson and Marcelo F. Vela  Diagnosis and Management of 
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7. Florian Rieder , Piero Biancani , Karen Harnett , Lisa Yerian , Gary W. Falk. Inflammatory 
mediators in gastroesophageal reflux disease: impact on esophageal motility, fibrosis, and 
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8. Kohata Y, Fujiwara Y, Machida H, Okazaki H, Yamagami H, Tanigawa T, Watanabe K, 
Watanabe T, Tominaga K, Wei M, Wanibuchi H, Arakawa T. Role of Th-2 cytokines in the 
development of Barrett's esophagus in rats. J Gastroenterol. 2011 Jul;46(7):883-93.   
9. Zhong YQ, Lin Y, Xu Z. Expression of IFN-γ and IL-4 in the esophageal mucosa of patients 
with reflux esophagitis and Barrett's esophagus and their relationship with endoscopic and 
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10. Motoyasu Kusano et al.  Rapid communication Development and evaluation of FSSG: 
frequency scale for the symptoms of GERD. J Gastroenterol 2004; 39:888–891. 

 
 
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11. Orosi P. Studies of phagocytic and killing activities of alveolar macrophages in patients with 
sarcoidosis. Lung. 1993; 171 (4): 591-593. 
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Baeten DL. Systematic validation of specific phenotypic markers for in vitro polarized human 
macrophages. J Immunol Methods. 2012 Jan 31;375(1-2):196-206. 
13. F.O. Martinez, A. Sica,  A. Mantovani, M.  Locati.  Macrophages activation and polarization 
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14. E.J.F. Vereyken, P.D.A.M. Heijnen, W. Baron,  et al. Classically and alternatively activated 
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specific CNS cell types. Journal of Neuroinflammation. -  2011. – Vol. 8. – P. 58 
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19. V. Shilling.  Blood picture and its clinical // Translation from German by S. Freidberg and A. 
Kranzfeld Editoe – E. Fromgold. — Мoscow: Gosmedizdat, 1931. – 412 p. (in Russian) 
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Liss. 2000, 565 p. 

 
 
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21. F. O. Martinez, L. Helming, R. Milde et al., “Genetic programs expressed in resting and IL-4 
alternatively activated mouse and human macrophages: similarities and differences,” Blood, 
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activation in fish Developmental and comparative Immunology pp. 1246-1255 
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injury in the presence of duodenal reflux.Surgery. 2012 Mar;151(3):382-90. doi: 
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13.0  APPENDIX A: FSSG