Professional Documents
Culture Documents
آخر واحد
آخر واحد
FINCTION
SBS 320
December 2018
University of Bahrain
Project Title:
Invitation:
This project is being carried out by a group of students from College of Health Sciences,
Alyaa Ali, Maryam Rashed, Maheen Abdelrahim, Hagar Nabil and Mariam Abdullah
Purpose of Study:
Before you decide to participate in this study it is important that you understand why the
research is being done and what it will involve. Please read the following information
carefully. Please ask the researcher if there is anything that is not clear or if you need more
information.
The purpose of this study is to investigate the changes in neutrophil function among type 2
diabetic patients.
Study Procedure:
In this study, a 5ml blood sample will be collected from each participant for laboratory
examination.
There will be no financial imputation to you for your participation in this study. However, we
hope that the information obtained from this study may help you to increase the awareness of
3
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
the complications that you may face as a diabetic patient and how to avoid it. On the other
Voluntary Participation:
Your participation in this study is voluntary. It is up to you to decide whether or not to take
part in this study. If you decide to take part in this study, you will be asked to sign this
consent form.
Confidentially:
Note that your personal information collected for this study will be anonymous. Participant
Note: If you have any questions you are free to ask anything regarding this study at any stage.
CONSENT
I have read and I understand the provided information and have had the opportunity to ask
chronic disease characterized by high blood glucose levels. Diabetes occurs mainly either due
primary function is to control blood glucose levels, which is achieved by transferring glucose
from the bloodstream to the body cells where it is converted to energy) or when there is an
impairment in the utilization of insulin by the body cells (WHO 2017). As a result, glucose
starts to build up in the blood vessels leading to hyperglycemia, which in turn may cause a
There are three main types of diabetes, Type1 (Autoimmune), Type2, and gestational
diabetes. Type 1 diabetes is also known as juvenile diabetes, early onset diabetes, or insulin-
dependent diabetes and characterized by a lack of insulin. This type accounts for 10% of all
Type 2 diabetes is the most common form of diabetes and was previously called an adult-
onset or non-insulin dependent diabetes. However, this type of diabetes arising either when
body cells are unable to utilize existing insulin properly or when insulin is produced
insufficiently. It usually develops in adults aged over 45 years, but can also occur in other age
groups such as children, adolescents, and young adults. Nearly 90-95% of all diabetes cases
Gestational diabetes is another form of diabetes that occurs during pregnancy and
characterized by high blood glucose above the normal range and below the diagnostic value
of diabetes. This group is at high risk to develop type 2 diabetes (WHO 2017).
Several complications are associated with type 2 diabetes involves short-term complications
syndrome (HHNS) are the major short-term complications faced by diabetic patients. On the
other hand, long-term complications are subdivided into microvascular and macrovascular
5
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
complications. As mentioned before, damage that occurs in tiny blood vessels
macrovascular complications are a heart attack, stroke, and peripheral vascular disease. In
Weintrob, A.C., and Sexton, D.J. (2009) reported/noted that “diabetics receive treatment for
infections more often than non-diabetics”. Recently, several epidemiological studies have
shown that type 2 diabetics may experience changes in neutrophils function, which
contributes to increased susceptibility and severity of infections. Neutrophils are the most
abundant type of white blood cells (~50-70%) in humans and play an essential role in the
body. It is considered as the first-line defense that migrates to the site of the infection to
ingest the microorganisms which is achieved by releasing a set of enzymes which in turn
Neutrophils play an essential role to protect the host from infectious agents and it is
important cells because their ability to migrate to varies areas in the body. When the foreign
antigens (such as bacteria) entering to the body tissue, it is stimulates the chemotaxis process
which results in the release of chemokines that attract circulating neutrophils to the
endothelium. Then, several receptors expressed on the endothelial surface (E-selectin) to bind
with neutrophil receptors (L-selectin) to allow rolling of neutrophil over the endothelial
surface. After rolling, neutrophil activates their integrin receptors allowing them to adhere to
endothelial I-CAM receptors which initiate the migration of neutrophil from circulation into
the affected tissue (diapedesis) with the help of PECAM-1 receptor (a receptor present on the
endothelial surface). After neutrophil enter the tissue, the most important neutrophil function
will begin by killing the organisms through the phagocytosis process. First of all, PRRs
on many microorganisms. Then, the plasma membrane of the neutrophil extended around the
foreign body and start to engulf it. After that, the engulfed particles are surrounded by a
phagosome to form a phagolysosome. As a result of this fusion, the lysozyme will release
hydrolytic enzymes which caused an acidic environment in the sac that lead to a destruction
of the microbes. Finally, the cellular waste products will be discharged from the cell through
exocytosis process. There are two mechanisms neutrophils used in order to destroy the
microbes after engulfment either by used oxygen-dependent mechanism that depends on the
Oxygen-dependent mechanism start when NADPH (One of the most significant components
of neutrophils which is scattered in plasma membrane and cytoplasm during resting state)
moves from plasma membrane to phagosome membrane and reduced to NADP and H +¿¿ by
an oxidation process. At the same time, this step will lead to oxidation of O2 into O2
−¿ ¿
(superoxide anion). After that, superoxide anion will react with H +¿¿ to produce H 2 O2
(Hydrogen peroxide) but this molecule is not very efficient as bactericidal agent, so in the
neutrophil) and Cl−¿¿(Chlorine) hydrogen peroxide will be converted to hypochlorite and this
The other mechanism is the oxygen-independent mechanism. This mechanism is not effective
as the oxygen-dependent ones and it has four main types. The first one uses cationic peptides
that damage the bacterium's membrane. The second type uses lysozymes that break down
peptidoglycan. The third type uses lactoferrins, which are a protein that prevents bacteria
from needed iron and present in neutrophil granules. The last type uses hydrolytic enzymes
7
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
and cathepsin G in which these enzymes cause damage to microbial membranes. The global
prevalence of diabetes has been steadily increasing over the past few decades from 4.7% in
1980 to 8.5% in 2014 (WHO 2017). In addition, the highest prevalence in Middle East and
North Africa region are mainly found in Saudi Arabia (18.5%), Bahrain (16.2 %), UAE
Literature Review
PubMed, Google Scholar, and MEDLINE were searched to identify studies published
between 2001 and 2013 reporting the relationship between type 2 diabetes and neutrophil
function. There was a study done by McManus, Bloodworth, Prihoda, Blodgett, and Pinckard
with extent of hyperglycemia). The purpose of this study was to determine if there is a
Through the study, researchers used some agonists such as calcium ionophore (A23187),
found that neutrophils dysfunction in diabetes patients occurred only with stimuli initiate
signal transduction through GPCR (agonists acting via G-protein-coupled receptors such as
fMLP and PAF) and this deficit inversely correlated with glycemic control. Thus, they
conclude that neutrophils function are progressively decreased as blood glucose levels are
increased, as well as, there were significant reductions of LTB4 production and lysosomal-
enzyme secretion.
One of the studies was carried out in 2004 by Gorudko et al. in order to investigate the
functional activity of neutrophils during type 2 DM and coronary heart disease (CHD),
particularly the role of the myeloperoxidase enzyme in the development of the oxidative
8
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
stress. In this study, the researchers studied peroxidase activity of myeloperoxidase, plasma
catalase activity, and levels of TBA-reactive lipid peroxidation products and oxidized
glutathione enzyme in three groups (healthy individuals, type 2 diabetic patients and type 2
diabetic patients with symptoms of CHD). Neutrophils have been stimulated to produce
a potent polymorphonuclear leukocyte (PMN) chemotactic factor. At the end of the study, the
researchers found that there was a marked increase in myeloperoxidase activity in patients
in the prevention of oxidative stress, its increased activity serve as an additional indicator of
oxidative stress in patients with DM. This, in turn, confirms the fact that patients with type 2
mainly in these cells and play a key role in the performance of its function.
Impaired chemotaxis, defective phagocytosis, and increased production of free radicals are
problems have been reported in diabetes mellitus. A respiratory burst that occurs via
activating the NADPH oxidase is one of the most important steps to stimulate phagocytosis
and thus kill bacteria. (Nicotinamide Effects Oxidative Burst Activity of Neutrophils in
Patients with Poorly Controlled Type 2 Diabetes Mellitus) is another study conducted in 2004
by Osar, Samanci, Demirel, Damci, and Ilkova. In this study, researchers hypothesized that
nicotinamide may restore the impaired oxidative burst capacity of neutrophils in diabetic
patients by increasing the NADH content as an electron donor and possibly through NADPH
oxidase activity. In order to examine this hypothesis, these experts divided the diabetics into
groups where one group was given nicotinamide and the other group received placebo for one
month. Eventually, scientists found that the oxidative burst activity in patients receiving
nicotinamide was greater when compared with the placebo group and the difference was
with the decreased activity of NADPH oxidase, which in turn are considered the key
elements used in the production of H 2 O2 and other oxidizing radical (Ex, oxidized halogens).
Diabetes mellitus patients with poor glycemic control was a study done by Lin et al. in 2005.
According to this study, the researchers presented that patients with liver abscess and septic
addition, 93% of endophthalmitis patients and 75% of liver abscess patients due to
K.pneumoniae were suffering from type2 DM. This study was done on three groups, in which
two groups had type 2 diabetes but one group with good glycemic control and the other group
with poor glycemic control and the third group was healthy control. The purpose of this
serotypes K1/K2 and non- K1/K2 K. pneumoniae among type 2 diabetes patients (with either
good or poor glycemic control) with healthy individuals by using Fluorescence technique
(fluorescein isothiocyanate) and electron microscopy. The result showed that the neutrophil
phagocytosis of K1 and K2 sera was low in patients with type 2 diabetes comparing to
healthy individuals. On the other hand, no different in phagocytosis was observed in non-K1
and K2 sera. Additional observation showed that diabetes patient with good glycemic control
has normal phagocytes activity. The researchers conclude that the major factor affecting
Another experimental study was done by Alba-Loureiro et al. in 2007 to examines the
relationship between biochemical metabolism and neutrophil function in rats with type 2
diabetes. The researchers studied the maximum activities of hexokinase enzyme (glycolytic
pathway) and citrate synthase enzyme (Krebs cycle) of neutrophil metabolism. At the end of
the study, they demonstrated that neutrophils from diabetic rats exhibit impairment in the
10
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
utilization of glutamine and glucose thus insufficient ATP synthesis and they conclude that
Shetty, Thomas, and Ramesh in 2008 made a study with a title of (Comparison of Neutrophil
Functions in Diabetic and Healthy Subject with Chronic Generalized Periodontitis). The aim
of this study was to evaluate the neutrophil function in 15 diabetic patients with chronic
generalized periodontitis. In order to study the neutrophil function, the researchers evaluate
(bacteria that cause periodontitis) by diabetic neutrophils relative to healthy and matched
control. At the end of the study, they observed significant depression in numbers of diabetic
production, and impaired phagocytosis. So the result explained that chronic generalized
In this study, the researchers evaluate the mitochondrial oxidative stress, levels of nuclear
DNA damage and apoptosis in peripheral blood neutrophils isolated from type 2 diabetes
mellitus patient (T2DM). For participation process, the diabetic participant was divided into
two groups: early and late onset of T2DM patients, and both participants were required to
Hemoglobin (HbA1C) previous assay results with no more than 1% variation. However, they
observed an elevation of mitochondrial oxidative stress in both early and late diabetes with a
Prevalence
11
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
Globally
The global prevalence of diabetes shown that the number of people with diabetes has
risen from 108 million in 1980 to 422 million in 2014, 422M are mostly adults over 18 years
old.
The report of WHO (2016) shown that the largest population with Diabetes disease are
present in South-East Asia and Western Pacific Regions. In few decades the same report has
shown that the numbers of Diabetes are raises rapidly due to same reasons, for example,
increase the age average of the population, population growth and increase in the prevalence
The mortality of people with diabetes was 1.5 million in 2012, it was conceded the eighth
leading cause of death during that years. Almost most of the death cases in diabetes are due to
high blood glucose level. The great mortality case due to the high glucose level occurs in
12
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
upper-middle income countries (1.5 million) and the lowest number in low-income countries
(0.3million).
Although Type 2 Diabetes seen mostly in adult and elderly people, now it’s arise in young
and children. There are no data on the true incident about the type 2 patient because most of
the time it is undiagnosed. The report from seven countries found that 24%-62% of people
with diabetes are not diagnosed and not treated. (WHO 2016).
Regionally
Recently, the prevalence of diabetes has been increased significantly in the Middle East
and North Africa (MENA) region. According to the International Diabetes Federation (IDF)
in 2017, almost 38.7 million adults aged 20-79 years were found in the MENA region. Nearly
to half (49.1%) of these cases were undiagnosed. More than two-thirds (67.3%) of adults with
MENA region becomes one of the highest rates of diabetes in the world. In addition, the
prevalence of diabetes has demonstrated a growing burden, and among the three types of
diabetes, type 2 diabetes is predominating while type 1 diabetes and gestational diabetes are
less common. However, the Gulf region has the highest prevalence among the MENA region,
four of the Gulf countries (Saudi Arabia, Bahrain, Kuwait, and United Arabic Emirate)
reporting the prevalence of equal to or more than 15% while Oman and Qatar with a
prevalence of less than 15% (IDF, 2017). The reason of increasing the prevalence in the Gulf
region is their recent societal changes with an elevation of economic development and growth
associated with decreased infant mortality and increasing life expectancy, resulting in a huge
change in the lifestyles, diet and physical activity in their population. However, Saudi Arabia
ranked first in the MENA region with a prevalence of 18.5% in 2017, while Yemen ranked
Bahrain has experienced a marked and rapid development in its socio-economic status,
leading to significant changes in lifestyle and patterns of health and disease. There has been a
sharp decline in infectious diseases and a rise in genetic diseases, including diabetes, which
has become one of the most common health problems in the kingdom.
According to the International Diabetes Federation, 5 out of 19 countries in the Middle East
and North Africa (MENA) with the highest prevalence of type 2 diabetes are the Gulf States,
including Bahrain, which ranks second where the prevalence rate reached 16.2 %. In 2016,
the number of diabetes cases among Bahrainis was 52,806 (Ministry of Health 2016). In
2017, this number has increased sharply, making the total number of diabetes cases become
Bahrain has witnessed a significant increase in diabetes mortality. Diabetes mortality rate was
10% in 2012, but this percent gradually rose to become 15% by the end of 2013 (Ministry of
Health 2012,2013). The probability of premature death from one of the non-communicable
14
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
diseases (NCDs), including diabetes is 13%, which means nearly one out of five adults die
Obesity, physical inactivity, tobacco use, and an unhealthy diet have played an important role
in increasing diabetes among Bahrainis. As a result, recent estimates suggest that diabetes
prevalence will increase by more than two-fold in Bahrain in the coming two decades,
Lab procedures
One of the most important tests to detect neutrophils function is nitroblue tetrazolium test
(NBT). NBT is a qualitative test that used to measure the ROS production particularly
chemical called nitro blue tetrazolium is added to white blood cells in the lab to check
whether there is change on its color or not. Normally, neutrophil changes the colorless
compound of NBT into dark blue aggregation and this indicates that cells should be able to
kill bacteria and protect the body from infections. But If there is no change in color when
NBT is added this indicates a reduced in ROS production due to NADPH oxidase defect and
Another test is done in the lab in order to asset neutrophilic function is Myeloperoxidase
(MPO). MPO enzyme is one of the most abundant proteins present in the neutrophils, it is
encoded by the MPO gene on chromosome 17, stored in the azurophilic granules and released
when neutrophils are stimulated. MPO is involved in microbe clearance by neutrophils both
intracellularly (via the production of Hypochlorous acid (HOCl) ) and extracellularly (via the
release of NETs). MPO oxidizes tyrosine to tyrosyl radical using H 2 O2 as an oxidizing agent.
Hypochlorous acid and tyrosyl radical are cytotoxic so they are used by neutrophil to kill
is a test used to measure alkaline phosphatase enzyme and tells how much of this enzyme is
presented in neutrophils. A blood sample is collected in (sodium or lithium heparin) tube then
separation of the neutrophils from the rest of the WBCs after that a blood smear is prepared
in order to stain and examine, the test involves counting 100 neutrophils (segmented and
band forms only ) then neutrophil leukocytes are scored from 0 to 4+ on the basis of the
intensity of the precipitated dye in their cytoplasm. The values of the 100 cells are added and
methodology:
16
TYPE 2 DIABETES AND ITS IMPACT ON NEUTROPHIL
References
Alba-Loureiro, T. C., Munhoz, C. D., Martins, J. O., Cerchiaro, G. A., Scavone, C., Curi, R.,
1044.
Gorudko, I. V., Kostevich, V. A., Sokolov, A. V., Shamova, E. V., Buko, I. V.,
medicine, 154(1), 23-26.
IDF Middle East and North Africa. (2017). Retrieved from https://www.idf.org/our-
network/regions-members/middle-east-and-north-africa/welcome.html
Khan, S., Raghuram, G. V., Pathak, N., Jain, S. K., Chandra, D. H., & Mishra, P. K. (2014).
Lin, J. C., Siu, L. K., Fung, C. P., Tsou, H. H., Wang, J. J., Chen, C. T., ... & Chang, F. Y.
McManus, L. M., Bloodworth, R. C., Prihoda, T. J., Blodgett, J. L., & Pinckard, R. N. (2001).
Shetty, N., Thomas, B., & Ramesh, A. (2008). Comparison of neutrophil functions in diabetic
of Periodontology, 12(2), 41.
Weintrob, A. C., & Sexton, D. J. (2009). Susceptibility to infections in persons with diabetes
http://www.who.int/mediacentre/factsheets/fs138/en/