A Christian’s relationship with God is vital to a Christian walk but other relationships and friendships require a reflection on the Christians personal walk with the superior being. Summary on biblical friendships Relationships gain more strength through proximity. The physical presence of friends is a source of joy and strength to a Christian. Distance makes friendship more difficult. For example, according to II John 12, John knew that he would be happier if he went to his own people and speak to them face to face (Dietrich B. 19).

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Runninghead:INDUCEDHYPERTENSION
Induced Hypertension
Student’s name
Institution

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Key points summary (150 word limit)
 Hypertension is a long-term medical condition that is a result of an increase in blood
pressure.
 Similar kinds of literature were looking into monocytes, neutrophils and different types
of immune cells and its relation to hypertension have shown that both T cells and
monocytes increased during hypertension.
 In this study, we show that the numbers of neutrophils did not significantly increase, but
the number of monocytes increased significantly. An increase of monocytes have vital
importance in the immune system. This is because they give the immune system the
ability to destroy invaders, while still facilitating healing and consecutively repair of
worn out body cells.
 Myelopoiesis is generally the term used in bone marrow production and all cells in it, that
is, all blood cells. In a narrower view, it is the regulated formation specifically of
myeloid, myelocytes and monocytes.
Abstract (5%) (250-word limit)
Hypertension is a long-term medical condition in which blood pressure in the arteries elevates.
Untreated and long-term hypertension can cause a major risk factor for coronary artery disease,
stroke, heart failure, kidney disease, and other vascular diseases. This experiment focuses on the
immune cells and how they cause hypertension. This experiment aimed to examine the role of
monocytosis in Angiotensin II-induced hypertensive mice. We hypothesized that monocytosis
plays a more vital role in hypertension in comparison to the lymphoid derived stem cells. Over

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the years, there are much literature supporting the information associating with immune cells and
hypertension. In our experiment using similar methods, we aim to determine if Ang II-induced
hypertension is associated with monocytosis. We used 12 mice and divided them randomly into
two groups, control and experimental. The experimental group received ANG II whilst the
control group received saline daily. A non-invasive tail-cuff method is used to measure blood
pressure before and after the experiment. Flow cytometry was used to isolate blood leukocytes
and bone marrow. Monocytes from the mice were then stained with antibodies with fluorophores
attached to them. We hypothesized that experimental hypertension is associated with
monocytosis which we believe promotes an inflammatory response in hypertension. The finding
of this study is a significant increase in myeloid cells that were predominantly monocytes.
Introduction (18%)
Hypertension is a significant medical condition is a result of an increase in blood pressure. It is
classified into three main categories, i.e., primary, secondary and resistant hypertension(Bhatt et
al.2014) Primary hypertension is most common and affects 90-95% of the population
(McManus et al. 2016) Secondary hypertension is resulted due to another overlapping condition.
Lastly, an individual with resistant hypertension concurrently uses four or more antihypertensive
agents of different classes to control blood pressure (Daskalopoulou et al. 2015). One of the
significant concerns associating hypertension is that the exact cause of it is still unknown.
Currently, Anti-hypertension drugs are used as the medication to treat hypertension. However,
one of the major problems when dealing with antihypertensive medicines is that these drugs only
target the symptoms and not the cause (De Franchi, 2015).

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In recent years, it has been shown that immune cells play a vital role in hypertension. Guzik
conducted a study in 2007, investigating the relationship between mice lacking T and B cells
(Rag mice) and their resistance to hypertension. The study showed that infiltration of T cells only
cause the development of hypertension and blood pressure is significantly higher in mice with
normal immune cells, whilst the rag mice showed lower blood pressure under angiotensin II
infusion. This suggests that T cells could increase blood pressure. Another study conducted by
Moore, JP, and colleagues (2015) study showed that by removing monocyte and macrophages in
the mice, had not resulted in experimental hypertension, which suggests monocyte plays a role in
hypertension.
The importance of what we will study is to understand the relationship between immune cells
such as monocytes and how they play a role in hypertension. Monocytosis is the increasing
number of monocytes circulating in the blood. Monocytes are generated from the bone marrow
where hematopoietic stem cells differentiate into myeloid progenitors cells (MPCs), MPCs then
differentiate into monocytes and enters the bloodstream through a process called myelopoiesis
(Schupp, J et al. 2017). Dragoljevic D and colleagues (2018) study shows that myelopoiesis has
been associated with inflammatory diseases such as atherosclerosis. The study used a flow
cytometry plot showing that mice with atherosclerosis have an increase in monocytes compared
to the control. Furthermore, it was seen that an increase in the number of myeloid progenitor
cells is detected in the bone marrow. We aim to determine if ANG II-induced hypertension is
associated with monocytosis and our hypothesis is the experimental hypertension is associated
with monocytosis which we believe promotes an inframammary response in hypertension.

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Methods (10%)
Several studies have used this model in conducting their research, in this experiment, there were
a control and an experimental group. Six mice aged 8-10 weeks were recruited for the
experimental group, these mice were then anesthetized with inhaled-, and following the
Angiotensin II-infused Mice Model of hypertension used in Guzik’s study, an osmotic minipump
will be implanted. This ensures subcutaneously delivers Angiotensin II (ANG-II) at
0.7mg/kg/day. In contrast, the control group received 0.9% saline + 0.01% acetic acid daily.
Mice were randomly allocated to the groups by a flip of a coin. The experimental group was
given ANG II for two weeks, and after two weeks, the mice were killed by inducing CO2, and
their blood samples were collected. The measurements of blood pressure were taken on Day 0
and Day 14 of the experiment via a non-invasive tail cuff method using volume pressure
recording. (CITE). From the blood sample, immune cells were extracted and labeled with
fluorophore-conjugated antibodies and then analyzed on a Cytoflex LS flow cytometer
(Beckman Coulter).
On the other hand, flow cytometry is used to collect bone marrow and blood leukocytes. Red
blood cells will firstly be lysed using ammonium chloride as the lysis buffer. The process of flow
cytometry proceeds with staining the blood with antibodies with fluorophores attached to them,
to different proteins such as Ly6C and CD 11b. Monocytes will be tainted with Ly6C+ and CD
115+. CMPs with CD34+ and FcgR III/II+Hi. GMPS with CN34+ and FegR III/II+ and lastly
MEP with CD34- and FegR III/II-. First, the cells will be in a tube and then labeled with
antibodies with fluorophores attached. Following this, the cells will incubate at room temperature
for 25 minutes and then washed, before being put on the flow cytometer for analysis. First, the

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cells were dissolved in mild surfactant which dissolved cell membranes and thus enhanced
permeability.
Figure 1. Expression of markers on the combined data on the expression of markers on the cells
analyzed.
An unpaired T-test conducts our experiment for flow cytometry, and a two way repeated
measures ANOVA for blood pressure. P<0.05 will be considered statistically significant

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Results (10%)
The results showed that the number of T cells did not change significantly between the
experimental and control groups. Therefore the results have shown that T Cells does not increase
during hypertension. It is shown that during hypertension there is an increase in myeloid cells. P
value in the unpaired t-test was significantly different as p=0.0353 / p<0.05.
Figure 2. Illustrates the different of BP from day 0 to day 14 indicates that the ANG II mice were
actually hypertensive.
Baseline Day 14
100
120
140
160
180
SystolicBP(mmHg)
Vehicle
Ang II *
*

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Figure 3. Illustrates a comparison of lymphoid cells between the vehicle and the experimental
group. All the three graphs i.e. 3A, 3B and 3C indicate that the proportion of T cells (CD8+ &
CD4+) remained more or less the same in both the experimental and vehicle group.
Figure 4: Illustrates the proportion of immune cells between the vehicle and the experimental
group. Figure 4A represents an increased proportion of myeloid cells in the experimental group
in comparison with the vehicle group. Figure 4B and 4C, both indicate a dramatic increase in the
proportion of monocytes, i.e. LY6CHI and LY6CLO respectively in the experimental group.
Figure 4D illustrates that the neutrophils proportion were not significantly different in both the
vehicle and the experimental group.

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Discussion (30%)
The main findings of the study was there is an increased amount of monocytes and myeloid
progenitor cells in blood during hypertension in the ANG ii induced mice compared to the
controlled mice. These findings were consistent with the literature. The increase of Ly6CHi and
Ly6CLo shown in Figure 4B and C is associated with hypertension. In the past years, studies
have shown that when Ly6Chi increased in blood, monocytosis increases, this indicates that
when there is a site of inflammation, these Ly6Chi monocytes will attract and infiltrate that site.
It will then differentiate into macrophages. Hence causing an inflammatory response. Therefore,
the results showed monocytosis in hypertension which supports our aim and hypothesis.
We also observed an increase in myeloid progenitor's cells in hypertensive mice shown in figure
4A, which suggested myelopoiesis is present. Another finding of our study is that immune cells
such as the T cell and Neutrophils shown in Figure 4 are not significantly different between the
control and experimental group. This suggests that hypertension is associated with myelopoiesis
and monocytosis specifically. A factor that stood out from our study comparing to other
literature are the number of leukocytes. In conclusion, this experiment has proven that
monocytes play a role to hypertension. The aim of our experiment was to determine if ANG II
induced hypertension is associated with monocytosis and myeloid cells. The hypothesis is
supported as its shown to associate with monocytosis. A limitation is that we could not
investigate on myelopoiesis as we were unable to obtain the bone marrow. Future studies should
involve bone marrow to confirm that myelopoiesis is associated with hypertension.

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