Clinical trial NCT04223479
Conditions: Ulcerative Colitis
Keywords: Ulcerative Colitis immune system Inflammatory markers Probiotics
Sponsor: University of Jordan
Start date: 2020-01-15; Completion date: 2022-03-10
Enrollment: 30 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Ulcerative colitis (UC) is a chronic Inflammatory bowel disease (IBD) that most likely results from the interaction between various environmental and genetic factors. Using probiotics as an adjunct to medical therapy might be useful in the treatment of UC and improving the symptoms of the disease. The result of studies that investigate the role of Probiotics supplementation in improving the inflammatory response, immune response and life quality of patients with the UC is not conclusive. So, this study aimed to study the effect of probiotics on the response of inflammatory markers, immune response, and quality of life in patients with UC.

An interventional double-blind randomized clinical trial (RCT) design will be used in this study. Forty patients will be recruited and randomly assigned to the placebo group (n=20) to receive 3 times a day placebo capsules; and probiotics group (n=20), to receive 3 times a day probiotic supplement. The demographic data, anthropometric measurements, IBD Quality of Life Questionnaire and blood samples will be collected at baseline and after 6 weeks of follow up. Interleukin-6, interleukin-1,interleukin-10 IL-10, C-reactive protein, tumor necrosis factor-alpha and complete blood count (CBC) will be measured.

The results will approve or disapprove the beneficial effect of using probiotics as adjuvant therapy for UC patients to raise the immune system as well as improving their quality of life.

Detailed description:
To meet the objectives of the study, an interventional double-blind randomized clinical trial (RCT) design will be used in this study. A placebo group will be included in parallel with the treatment group in this trail. Forty patients (aged 35-65 years) who diagnosed with mild to moderately active UC will be recruited conveniently from the gastroenterology section, the IBD clinic at the Jordan University Hospital, Amman, Jordan. Patients who meet the inclusion criteria and agree to participate will be centrally randomized to probiotic supplementation group or placebo group using computer-generated random numbers, that balanced allocation to groups A and B: in successive blocks each containing 20 patients each stratified by gender. The duration of the intervention will be 6 weeks.

For the participants, the Jordan University Hospital setting will be utilized for data collection. The patients will be recruited over 12 months and all patients will be asked to sign a written informed consent before enrollment. The patients will randomly be assigned to the placebo group (n=20), to receive 3 times a day placebo capsules contain polysaccharides, without any viable probiotics matching the probiotic capsules in appearance, smell, and taste; and probiotics group (n=20), to receive 3 times a day probiotic supplement. The administration of supplements will be under the supervision of the treating physician. The blood sample will be collected at baseline and at the end of 6 weeks of follow up.

The demographic data of each subject will be collected such as; gender, age, body mass index (BMI), tumor location, malignant tumors stage, tumor differentiation, educational level, occupation, family history, smoking, dietary and physical activity. At baseline and end of the follow-up, IBD Quality of Life Questionnaire will be collected and blood sample tests will be withdrawn and the following biochemical variables will be measured: immunoglobulin G, immunoglobulin M, immunoglobulin A, interleukin-6 (IL-6), C-reactive protein (CRP), interleukin-1(IL-1), interleukin-10(IL-10), interleukin-12 (IL-12), Tumor necrosis factor-alpha (TNF-α) and complete blood count (CBC).

Arms: arm 1: In this intervention arm, the patients will receive oral viable capsules of probiotic contain (1\*10 10 colony-forming unit (CFU)/g) of lactobacillus (Lactobacillus rhamnosus , Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum) and bifidobacteria (Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium infantis) species three times a per day arm 2: In this intervention arm Placebo arm received three oral viable capsules daily, containing polysaccharides, without any viable probiotics matching the probiotic capsules in appearance, smell, and taste.
Interventions: intervention 1: Probiotic Formula Capsule intervention 2: Placebos
Intervention descriptions: intervention 1: The Probiotic will be coded in a specific label by a researcher who will not be in contact with the participants and administrated randomly based on gender in a double-blind manner. The patient will receive 2 bottles of the drug every 2 weeks and will be followed weekly for 6 weeks. intervention 2: The Placebos will be coded in a specific label by a researcher who will not be in contact with the participants and administrated randomly based on gender in a double-blind manner. The patient will receive 2 bottles of the placebo every 2 weeks and will be followed weekly for 6 weeks.

Protocol documents:
Prot_000.pdf: 
 
Study Protocol  
Effect of Probiotic Supplementation on the Immune System in 
Patients With Ulcerative Colitis in Amman, Jordan  
 
 
NCT04223479 
 
 
 
 
 
 
Date:11 September 2022 
 
 
The Objectives of the Study are: 
 
1- To determine the effect of Lactobacillus and Bifidobacterium probiotics supplementations 
on immune system response among patients diagnosed with pre-remission mild to moderate 
UC.  
2- To determine the effect of Lactobacillus and Bifidobacterium probiotics supplementations 
on inflammatory response among patients diagnosed with pre-remission mild to moderate 
UC. 
3- To determine the effect of Lactobacillus and Bifidobacterium probiotics supplementations 
on quality of life for pre-remission mild to moderate UC. 
 
 
The Research Problem: 
 
Ulcerative colitis (UC) is a chronic Inflammatory bowel disease (IBD) that most likely 
results from the interaction between various environmental and genetic factors (Van Assche et al., 
2010). UC is currently defined by continuous mucosal inflammation in the rectum and extended 
variables in the colon, without the existence of granulomas on mucosal biopsies (Dignass et al., 


 
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2012a). UC is a lifetime disease that is characterized by periods of remission and periods of 
relapse. The relapsing periods often presented with a combination of rectal bleeding, diarrhea, 
abdominal pain, malaise and weight loss, which are responsible for the decreased quality of life 
and for most of the disease burden (Dignass et al., 2012b; Levesque et al., 2015). Moreover, 
newly diagnosed UC patients have a 5-year risk of colectomy of 10–35% (Van Assche et al., 
2013), and ultimately, the extensive and persistent inflammation can increase the long-term risk of 
colorectal cancer (Van Assche et al., 2013). Worldwide, UC is reported to have an overall 
prevalence range from 7.6 to 245 cases per 10 0,0 0 0 persons/year and an incidence of 1.2–20.3 
cases per 10 0,0 0 0 persons/year (Ng et al., 2017; Feuerstein et al., 2019; Gajendran et al., 2019). 
For specific countries, the UC prevalence in the United States was 286 cases per 100,000 
populations (Kaplan and Ng, 2016), in San Marino was 311 per 100,000 populations (Piscaglia et 
al., 2019), and in Norway 505 of 100,000 populations (Ng et al., 2017). In Jordan, there have been 
no studies conducted on the prevalence of UC. However, the incidence of UC is growing in 
Jordan, with changes in the cultural behavior influenced by Westernization and now converted to a 
country with varied ethnicities (Omran and Barakat, 2016). 
 The Food and Agriculture Organization of the United Nations (FAO) and the World Health 
Organization (WHO) nutritional guidelines have defined probiotics as “live microorganisms when 
administered in adequate quantities confer a health profit to the host cell” (Araya et al., 2002; 
FAO/WHO,2013; Nolfo et al., 2013). Probiotics have been used in IBD and especially UC (Sartor, 
2004). The effect of using probiotics on UC has been studied in a variety of experimental 
conditions both in vitro and in vivo (Abraham and Quigley 2017; Guandalini and Sansotta, 2019). 
The rational explanation for using probiotics in UC is based on the hypothesis that intestinal 
bacteria might be associated with the pathogenesis of IBD in general, and UC in specific (Sartor, 
2004). Balance disturbance between the host’s genetic background, enteric microflora, and bowel 
mucosal immunity might be involved in tissue inflammation in UC (Naidoo et al., 2011).  


 
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The use of probiotics to enhance immune responses, attenuate inflammatory responses, improve 
the intestinal microbial environment and as conventional therapy for patients with IBD in general, 
and for UC in specific were researched (Mardini and Grigorian, 2014; Shen et al., 2018; Dargahi 
et al., 2019). However, results are still inconclusive and insufficient to recommend for or against 
the use of probiotics either alone or in combination with standard therapy for UC (Mardini and 
Grigorian, 2014; Shen et al., 2018; Dargahi et al., 2019). Additionally, in Jordan, no studies have 
been conducted on the uses of probiotics for UC patients. Therefore, this study aims to evaluate 
the effect of probiotics supplementations on the immune system and inflammatory responses 
among mild to moderate UC patients pre-remission in Amman, Jordan. 
The importance of the Study:  
The importance of the present study lies on establishing information that either validates or 
invalidates the effectiveness of probiotics supplementation intervention in reducing inflammatory 
markers and enhancing the immune system response for patients with pre-remission mild to 
moderate UC. Moreover, the results could provide additional evidence to the beneficial effect of 
using probiotics as adjuvant therapy for UC patients to elevate their immune response and reduce 
the inflammatory marker during reaching the remission status. 
 Literature Review: 
Ulcerative Colitis (UC): 
In the 21st century, Inflammatory bowel disease (IBD) is a global disease that includes ulcerative 
colitis (UC), Crohn’s disease (CD), and indeterminate colitis (IC) (Bamias et al., 2012; Ng et al., 
2017). In contrast of Crohn's disease, which are healthy parts of the intestine mixed in between 
inflamed areas that can occur in all the layers of the bowel walls (Dignass et al., 2012a; Gajendran 
et al., 2019). Ulcerative colitis (UC) is a continuous inflammation of the colon, lifelong, chronic 


 
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IBD that only affects the inner most lining of the colon and most likely results from the interaction 
between genetics and various environmental factors (Dignass et al., 2012a; Gajendran et al., 
2019). UC is defined as a chronic inflammatory condition resulting in a continuous mucosal 
inflammation starting with mucosal inflammation of the rectum and extending proximally to the 
colon without the existence of granulomas on mucosal biopsies, characterized by a period of 
remission and period of relapse (Dignass et al., 2012a; Gajendran et al., 2019). 
UC Prevalence: 
Ulcerative colitis (UC) is the most common IBD worldwide (Gajendran et al., 2019).  It can 
develop at any age but has an incidence peak occurs in the 2nd or 3rd decades of life (between 10 
and 29 years of age), followed by a second peak between 50 and 80 years of age (Feuerstein et al., 
2019). UC is more common in developed countries including North America and Western Europe, 
and recently there has been increased incidence in Asia (Feuerstein et al., 2019; Gajendran et al., 
2019). Additionally, the incidence of UC increased in newly industrialized countries whose 
societies have become more westernized (Ng et al., 2017). The overall incidence of UC is reported 
to be 1.2–20.3 cases per 10 0,0 0 0 persons/year, while the prevalence is reported to be 7.6–245 
cases per 10 0,0 0 0 persons/year (Ng et al., 2017; Feuerstein et al., 2019; Gajendran et al., 2019). 
A systematic review of population-based studies conducted on the incidence and prevalence of 
inflammatory bowel disease worldwide in the 21st century as well as other studies on the 
prevalence of UC, reported that the prevalence of UC was 286 cases per 100,000 populations in 
the United States (Kaplan and Ng, 2016), 311 of 100,000 in San Marino (Piscaglia et al., 2019), 
505 of 100,000 in Norway, and a low of 6.67 per 100,000 in Malaysia (Ng et al., 2017). Also, they 
found that the prevalence of IBD (UC) exceeded 0·3% in North America, Oceania, and many 
countries in Europe (Ng et al., 2017). 


 
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 In Jordan, there is very scare information about the UC disease and the information about its 
prevalence and incidence are is not known (Omran and Barakat, 2016). However, the UC is not 
rare and occur among all age groups, with a peak incidence in the second and third decades 
(Ghazzawi and Al-Marayat, 2007; Omran and Barakat, 2016).  
UC Risk Factors and Clinical Manifestations: 
The key risk factors associated with the development of UC are genetics, environmental factors, 
autoimmunity, and gut microbiota (Dignass et al., 2012a; Gajendran et al., 2019). The genetic 
predisposition plays a role in 8–14% of UC cases where patients have family history of UC 
(Gajendran et al., 2019). The environmental risk factors associated with the development and 
severity of UC are ethnic and racial differences, which have reported to be more related to 
environmental influences, food habits and lifestyle rather than the true genetic differences 
(Gajendran et al., 2019). Other environmental risk factors include smoking, age, and diet 
("Western" style diet) differences (Dignass et al., 2012a; Gajendran et al., 2019). Intestinal 
microbiota populations composition alteration associated with dysregulation of the immune 
response to bacterial antigens (Lepage et al., 2011; Gajendran et al., 2019).  
The typical symptoms of UC are bloody diarrhea, abdominal pain, fecal urgency, tenesmus, and 
mucus in the stool (Feuerstein et al., 2019; Gajendran et al., 2019). The diagnosis of UC is made 
based on a combination of clinical features, Lab results, and endoscopic findings that show a 
continuous and diffuse colonic inflammation which starts in the rectum, as well as histological 
findings where biopsies of the colon documenting a chronic inflammation that confirm the 
diagnosis of UC (Feuerstein et al., 2019; Gajendran et al., 2019). 
UC Managements: 
The management of UC is multi-disciplinary and changing process, aiming mainly to induce 
clinical remission and then the maintenance of remission, which defined as normalization of bowel 


 
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movements and cessation of bleeding (Dignass et al., 2012b; Vanga and Long, 2018). The suitable 
treatment approach for each patient is personalized based on the activity, distribution (proctitis, 
left-sided, extensive1), pattern of disease (relapse frequency, course of disease, response to 
previous medications, side-effect profile of medication and extra-intestinal manifestations) and the 
age at onset and disease duration (Dignass et al., 2012b). Most patients are treated with 
pharmacological therapy to first induce remission and then to maintain a corticosteroid-free 
remission. There are multiple classes of drugs used to treat the disease (Vanga, and Long, 2018). 
However, the patients in which medical therapy fails or have developed dysplasia secondary to 
their long-standing colitis require surgical treatment (Ray and Sagar, 2017; Feuerstein et al., 
2019).   
In addition to the progression in the treatment of inflammatory bowel diseases (IBD) specially UC, 
adjuvant and alternative options are continually seeking and needed to enhance and improve the 
pharmacological treatment (Guandalini and Sansotta, 2019). These options include dietary 
modifications, food supplements, and, more recently, probiotics (Guandalini and Sansotta, 2019). 
Mucosal homeostasis or chronic inflammation are occurring as a result of the imbalance between 
beneficial and harmful intestinal microorganisms which known to be associated with IBD 
occurrences (Guandalini and Sansotta, 2019). The UC and IBD patient’s microbiota differ from 
healthy individuals, as it is characterized by a reduced diversity, reduced abundance of Firmicutes 
and Bacteroidetes, and increased the abundance of Enterobacteriaceae (Guandalini and Sansotta, 
2019). 
Probiotics  
As the demands for healthy and functional foods that promote health and prevent or cure illness 
increased during the last decades, probiotics have received attention in the field of self-care and 
complementary medicine (Shokryazdan et al., 2017a). The word “probiotic” comes from the 


 
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Greek words “pro” and “biotic,” meaning “for the life” (Shokryazdan et al., 2017a). In 2001, the 
Food and Agriculture Organization/World Health Organization (FAO/WHO) defined probiotics as 
“live microorganisms which, when administered in adequate amounts confer a health benefits on 
the host.”  (FAO/WHO, 2001). This concept was updated in 2013 to include the three main key 
aspects of probiotics: microbial, viable, and beneficial to health, to became “live microorganism 
that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 
2014). Microorganisms that are considered as probiotics should have several characteristics 
including resistance to gastrointestinal environment (low pH and bile salt), antimicrobial activity, 
multidrug resistance, and antioxidant activity (Biradar et al., 2008). The chief and most widely 
used probiotics bacterial species belong to the genera Lactobacillus and Bifidobacterium, which 
belong to lactic acid bacteria group (Ritchie and Romanuk, 2012; Sharif et al., 2018). 
Probiotics Health Benefits 
The human gastrointestinal tract (GIT) is a complex microbial ecosystem inhabited by more than 
400 bacterial species, which can be influenced by different factors, and one of the most important 
factors is the diet of the host (Holzapfel and Schillinger, 2002; Graf et al., 2015).  Recent scientific 
research showed that the deficiency or imbalance of intestinal microbiotate leads to some of the 
infections and disorders (Shokryazdan et al., 2017a; Sánchez et al., 2017). Probiotics have been 
considered as one of control strategies for several gastrointestinal disorders such as GIT infections, 
constipation, irritable bowel syndrome, inflammatory bowel disease (Crohn’s disease and 
ulcerative colitis), antibiotic-induced diarrhea, food allergies, and certain cancers such as 
colorectal cancer (Shokryazdan et al., 2017a; Sánchez et al., 2017).   
The health promoting benefits of probiotics include modulation of the immune system reducing 
colitis and inflammation, antioxidant activity, toxin-binding and detoxification activity, 
maintenance of mucosal integrity, decreasing incidence and duration of diarrhea, and regulation of 


 
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gut motility to control constipation or irritable bowel syndrome (Tannock, 1999; Orrhage and 
Nord, 2000; Zoghi et al., 2014). Additionally, probiotics may reduce allergy symptoms, improve 
nutrient absorption, alleviate symptoms of lactose intolerance, and produce beneficial compounds, 
such as vitamins, short-chain fatty acids (SCFAS), and conjugated linoleic acid (Shokryazdan et 
al.,2014; Isolauri et al., 2015; Onubi et al.,2015; Liu et al.,2017; Shokryazdan et al., 2017b).  
Regarding the safety, there is no evidence that consumption of lactobacilli probiotics causes any 
risk of infection greater than that associated with commensal strains and it is impossible to propose 
a risk of death for lactobacilli consumption (Borriello et al., 2003). 
Probiotics and Immune Response 
The protection of gastrointestinal health is an essential medical issue because 70% of the human 
immune system is placed in the intestine (Sharif et al., 2018). Probiotics help to enhance intestinal 
barrier function and maintain the integrity of the gut and other organs by secreting antioxidative 
and anticarcinogenic compounds that help in maintaining and supporting the immune system 
(Sharif et al., 2018; Dargahi et al., 2019). Probiotics stimulate the immune system function by 
producing anti-inflammatory cytokines; stimulating dendritic cells (DCs) to produce inflammatory 
cytokines interleukin-12 (IL-12) and regulatory IL-10; down-regulating IL-12 levels through 
surface layer proteins that interact; and stimulating the innate cells (Kanmani et al., 2013; Zhong 
et al., 2014; Witkowski et al., 2018). Furthermore, probiotics enhance the total numbers of T cells, 
natural killer (NK) cells, histocompatibility complex (MHC) class II+ cells, and CD4-CD8+ T 
cells and increase the production of cytokines, such as interferon (IFN)-γ, interleukin1-β (IL-1β) 
and tumor necrosis factor alpha (TNF-α) (Kanmani et al., 2013; Zhong et al., 2014; Dargahi et al., 
2019). 
Studies of animal and human subjects indicated that several isolates of Lactobacillus plantarum, 
Lactobacillus casei, Lactobacillus rhamnosus GG, and/or Lactobacillus acidophilus can mediate 
anticancer effects through different mechanisms, including maturation of dendritic cells, release of 


 
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probiotic-derived ferrichrome, or activation of natural killer cells (Hu et al., 2015; Cai et al., 2016; 
Konishi et al., 2016; Dargahi et al., 2019). Additionally, Lactobacillus mixture shifted the gut 
microbial community toward Prevotella and Oscillibacter, which are known to produce anti-
inflammatory metabolites that decrease Th17 polarization and enhance the differentiation of anti-
inflammatory Treg/Tr1 cells in the gut (Li et al., 2016; Dargahi et al., 2019). 
Probiotics Role in Ulcerative Colitis 
The uses of probiotics for UC have been explored under a variety of experimental conditions both 
in vitro and in vivo (Abraham and Quigley 2017; Guandalini and Sansotta, 2019). Studies revealed 
that using of probiotics as an adjunct to medical therapy might be useful in the treatment of UC 
(Shen et al., 2018; Guandalini and Sansotta, 2019). This beneficial effect of Probiotics may be 
explained through at least two mechanisms (Witkowski et al., 2018). First one, their ability to 
enhance the mucosal barrier function, which shown to promote the secretion of IgA and mucins 
(Kabeerdoss et al., 2010) and tight junction function (Karczewski et al., 2010; Kotzampassi and 
Giamarellos-Bourboulis, 2012). Secondly, through their interaction with the local immune system 
to enhance regulatory T cell responses, decreased the pro-inflammatory cytokines such as TNF-α 
and IL-1β and increased anti-inflammatory factor IL-10 (de Moreno et al., 2011; Zhao et al., 
2013), consequently improving the symptoms of UC, and this mediated through PI3K/Akt and 
NF-B signaling pathway (Shen et al., 2018; Witkowski et al., 2018). Bifidobacterium probiotic is 
one of beneficial microbiota that promotes antitumor immunity and prevents recurrence of UC 
(Ventura et al., 2014). Anther probiotic that studied and used for treating UC are E. coli Nissle 
1917, which widely used in Europe (Scaldaferri et al., 2016), Saccharomyces boulardii (Thomas 
et al. 2011; Limketkai et al., 2018) and lactic acid-producing bacteria ( L. plantarum, L. 
delbrueckii subsp. bulgaricus, L. casei, L. acidophilus, Bifidobacterium breve, B. longum, B. 
infantis, L. paracasei subsp. paracasei, Lactobacillus casei strain Shirota and Streptococcus 
salivarius subsp. Thermophilus) (Limketkai et al., 2018; Guandalini and Sansotta, 2019). 


 
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Probiotics also found to have an effect in inducing, maintaining and increasing the remission 
period in UC and this effect suggested to be also associated with the immunomodulator role of 
certain microorganisms (Tamaki et al. 2016). For example, in vitro studies, Lactobacillus casei 
strain Shirota found to have anti-inflammatory effect by inhibition of IL-6 and interferon c (IFN-c) 
synthesis in lipopolysaccharide (LPS)-stimulated murine chronic IBD cells (Matsumoto et al. 
2005), and Lactobacillus delbrueckii had anti-inflammatory effect by inhibition of NF-kB pathway 
in large intestinal lamina propria mononuclear cells (Santos Rocha et al. 2012). In vivo, 
Lactobacillus delbruekii and Lactobacillus fermentum supplementation, in UC patients undergoing 
treatment with sulfasalazine (an anti-inflammatory medicine) showed a reduction in several pro-
inflammatory parameters in the colon, such as IL-6 levels, tumor necrosis factor TNF-α and NF-
kB p65 expression, and leukocyte recruitment, in comparison with the placebo and medical 
treatment groups (Hegazy and El-Bedewy 2010). In another study where Bifidobacterium breve 
was administrated as a part of a symbiotic, the results showed a significant decrease in the 
inflammatory parameters and improvement in the clinical status evaluated by colonoscopy in 
patients with mild to moderate UC (Ishikawa et al. 2011). Moreover, administration of probiotic 
product Bifid Triple Viable, that contains Lactobacillus acidophilus, B. bifidum and 
Streptoccocus, for 2 months, lead to reduce the expression of IL-1b and increased the expression 
of IL-10 and immunoglobulin A (IgA) in the colon of UC patients (Li et al. 2012). 
Materials and Methods: 
Study Design 
An interventional double-blind randomized controlled trial (RCT) design will be used in this 
study. A placebo group will be included parallel with the treatment group in this study. Forty IBD 
patients with mild to moderately active UC aged 18-65 will be recruited conveniently from the 
gastroenterology section, the IBD clinic at the Jordan University Hospital and Al-Bashir Hospital, 
Amman, Jordan. Patients who meet the inclusion criteria and agree to participate will be centrally 


 
11
randomized to probiotic supplementation group or placebo group using computer-generated 
random numbers, stratified by age and gender. The duration of intervention will be 6 weeks.  The  
study scheme: 
 
The Interventions 
The probiotics and placebo treatments will be given at weekly basis in double blind manner for the 
six weeks. The patients in the probiotics group will receive 3 oral viable capsules of Jamieson 
Probiotic 10 Billion product, each of which contain (1*1010 CFU/g) of lactobacillus (Lactobacillus 
rhamnosus HA-111, Lactobacillus acidophilus HA-122, Lactobacillus salivarius HA-118, 
Lactobacillus paracasei HA-196) and bifidobacteria (Bifidobacterium breve HA-129, 
Bifidobacterium bifidum HA-132 HA, Bifidobacterium longum HA-135) species three times a day. 
Similarly, the placebo group will receive 3 placebo capsules containing maltodextrin three times a 
day. 
40  IBD patients with mild to moderately active UC who meet the inclusion criteria 
and agree to participate will be assigned into 2 groups: 
 
Data and blood 
samples collection 
 
Data and blood 
samples collection 
 
 Follow-up for 6 weeks
 
Supplementation group:  20 
Patients will be assigned to 
probiotic intervention 
Placebo group: 20 Patients 
will be assigned to placebo 
intervention 


 
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Human Participants:  
Inclusion and Exclusion Criteria: 
Inclusion criteria will be patients with age between 18 -70 years and diagnosed with UC 
established by colonoscopy and histology, and suffering from mild to moderate UC as defined by 
Modified Mayo Disease Activity Index (MMDAI, table 1) (score 3–9), the diagnoses will be done 
by a physician (Ng et al., 2010; Palumbo et al., 2016; Tamaki et al. 2016). Patients will be 
excluded if they had age <18 years, >70 years, pregnancy, planned pregnancy, breastfeeding, 
evidence of severe disease (MMDAI >10), concurrent enteric infection, use of antibiotics within 
the past 2weeks, change in dose of oral 5-aminosalicylic acid (5-ASA) within the past 4weeks, and 
use of rectal 5-ASA or steroids within 7 days before entry into the study, or the use of probiotic 
preparations during study, received any investigational medicines within 3months, and if they have 
significant hepatic, renal, endocrine, respiratory, neurological, or cardiovascular diseases were also 
excluded (Ng et al., 2010; Bengtsson et al., 2016; Tamaki et al. 2016; Palumbo et al., 2016).  
 
Ethical Approval: 
Palumbo et al., 2016.
 


 
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This study will seek approval by Research Ethics Committee at the University of Jordan and by 
the Institution Research Board (IRB) of Jordan University Hospital and Al-Bashir Hospital, 
Amman, Jordan. The participants will be provided with information consent containing a brief 
description of the study and written informed consent will be obtained from all participants before 
their interview and blood drawl (10ml) (Appendix 1, 2). Participants’ information will be treated 
confidentially. However, only the researcher (PhD student) will know participants’ names and she 
is the only one who will give them identification (ID) number. 
Personal Questionnaire: 
The personal questionnaire will contain questions related to age, gender, education, employment, 
family income/month, residency area, smoking status, family history of IBD, medications, 
previous and current health problems. As well as, questions about using probiotics and certain 
eating habits will be asked (Appendix3). Anthropometric measurements including weight 
(measured to the nearest 0.1 kilogram) and height (measured to the nearest 1 cm) will be taken 
from the existing medical record. Body mass index (BMI) will be calculated.  
Biochemical Measurements: 
The resercher will take the permission form the participants to draw blood sample (10ml) and 
perform biochemical specialized labs. Venous blood samples will be withdrawn by specialized 
medical laboratory technicians. Complete blood count (CBC) will be analyzed using standard 
precedure. Blood serum will be separated by centrifugation (3000 rpm for 10 min) after clotting at 
room temperature (27 °C) and stored at -18°C until analysis. Immunoglobulin G(IgG), 
Immunoglobulin M (IgM), Immunoglobulin (IgA), interleukin (IL)-6, IL-1, IL-10, C-Reactive 
Protein (CRP), and TNF-α will be analyzed using commercially available enzyme-linked 
immunosorbent assay (ELISA) kits.  


 
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Data Collection and Quality of Life Questionnaire 
Three parts package was used for collecting data that met the purpose of this study, including a 
personal questionnaire, physical activity questionnaire, and a short inflammatory bowel disease 
questionnaire (SIBDQ). A face-to-face interview technique was used as a method for data 
collection. Data on age, body weight, educational level, previous and current health problems, 
family history of IBD, and probiotic use were collected. The physical activity level was measured 
using a 7-day Physical Activity Recall (PAR) which was originally developed by Sallis et al., 
1985 (Sallis et al., 1958). For assessing the quality of life, a disease-specific, validated, and 
reliable Arabic version of the SIBDQ obtained from the McMaster short IBD questionnaire at 
McMaster University was used (Mahalli and Alharthi, 2017). The SIBDQ contains ten questions 
covering the four HRQOL domains (systemic, social, bowel, and emotional) which were 
calculated and scored. The response to each question of the ten was graded on a 7-point Likert 
scale ranging from 1 (worst aspect) to 7 (best aspect). The total SIBDQ scores range from 10 to 
70, with higher scores indicating better HRQOL (Mahalli and Alharthi, 2017). The summation of 
the responses to Q4, Q6, and Q9 was used for the bowel domain, the sum of the responses to Q2 
and Q3 was for the social domain, the sum of the responses to Q1 and Q7 was for the systemic 
domain, and the sum of the responses to Q5, Q8, and Q10 was for the emotional domain. The total 
score of SIBDQ was composed of the sum of the responses to all ten questions (Guyatt et al., 
1989; Mahalli and Alharthi, 2017).
 
Statistical Analysis: 
Statistical analysis will be done using IBM SPSS Statistics 20.0 software (version 20.0, IBM, lnc., 
Chicago, IL, USA). Mean ± standard deviation (SD) will be used to express the continuous data 
and percentage to express the categorical data. The categorical variables will be compared using 
the Chi square and the continuous variables will be compared using the one -way ANOVA. A 


 
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paired t-test will be used to assess within-group changes in outcomes from baseline to end of 
intervention. For all data, P< 0.05 will be considered statistically significant. 
References  
Abraham, B. P., and Quigley, E. M. (2017), Probiotics in inflammatory bowel disease. 
Gastroenterology Clinics, 46(4), 769-782. 
Araya M, Morelli L, Reid G, et al. (2002), Guidelines for the evaluation of probiotics in food. 
Joint FAO/WHO Working Group report on drafting guidelines for the evaluation of probiotics 
in food, London; (ON, Canada). ftp://ftp.fao.org/es/esn/food/wgreport2.pdf (Accessed 
19March, 2018). 
Bamias, G., Corridoni, D., Pizarro, T. T., and Cominelli, F. (2012), New insights into the 
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SAP_001.pdf: 
Effect Of Probiotic Supplementation on The Immune 
System in Patients with Ulcerative Colitis in Amman, Jordan 
By 
Lana Mustafa Agraib 
 
 
Supervisor 
Dr. Mohammed Isam Yamani 
 
 
Co-Supervisor 
Dr. Reema Fayez Tayyem 
 
 
Funded by the University of Jordan 
 
 
22 November  2023 
 
 
 NCT04223479 
 
 


Statistical Analysis 
 Statistical analyses were performed using Statistical Package for Social Science (IBM® SPSS® 
Statistics; Version 21.0, version for Windows, 2019, USA). Mean and standard deviation (SD) 
were calculated for the continuous variables. Frequencies and percentages were used to describe 
categorical variables. The frequency of participants in terms of categorical variables was 
compared using the X² test. To check the normal distribution of continuous variables, the Shapiro-
Wilk test was applied. Independent samples T-test was used to detect differences between placebo 
and probiotics groups in terms of continuous variables. A paired t-test was used to assess within-
group changes in biochemical parameters (IgG, IgM, IgA, IL-1, IL-6, IL10, TNF-α, and CRP) 
from baseline to the end of the study. A p-value < 0.05 was considered statistically significant. All 
outcomes were calculated by intention-to-treat (ITT) and perprotocol (PP) analyses. For ITT 
analysis, all randomized patients who took at least one tablet of study medication were included. 
For PP analysis all protocol violators (patients’ dropout or loss of follow-up) were excluded.