1. health and fitness
2. disease

Republic of Iraq
Ministry of Higher Education & Scientific Research
Baghdad University/College of Science
Department of Biotechnology
Immunological and Biochemical profile of
Alzheimer's Disease in a Sample of Iraqi
Patients
A Thesis
Submitted To the College of Science / University of
Baghdad in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy in Biotechnology/
Immunology
By :
Alaa Abd-Alhasan Hamdan Al-Ganzawi
B.Sc. (2006) College of Science / University of Thi-Qar
M.Sc. (2009) College of Science / University of Baghdad
Supervised by :
Dr. Alice K. Melconian
Professor
October- 2013
Dr. Ali H. Ad'hiah
Professor
Dhu-Al-Hijja- 1434
‫بسم اهلل انرمحه انرحيم‬
‫وَانهَّهُ خَهَقَكُمْ ثُمَّ يَتَوَفَّبكُمْ وَمِنْكُمْ‬
‫مَهْ يُرَدُّ إِنَىٰ أَرْذَلِ انْعُمُرِ نِكَيْ نَب يَعْهَمَ بَعْدَ عِهْمٍ شَيْئًب‬
‫إِنَّ انهَّهَ عَهِيمٌ قَدِيرٌ‬
‫صدق اهلل انعظيم‬
‫سورة اننحم اآلية {‪}07‬‬
Dedication
To who sets my steps on the beginning
of the way……my dear father
Alaa
Acknowledgement
I would like to thank Allah's for His care and support throughout my
life and especially through the accomplishment of this research.
I would like to express my profound thanks and sincere gratitude to my
supervisor Dr. Alice K. Melconian for her valuable scientific advice. I extend
my deep gratitude also to my supervisor Dr. Ali H. Ad'hiah for his scientific
guidance, support and encouragement that made it possible for me to
accomplish this study.
I should express my gratitude to the Head of the Department of
Biotechnology and to the Dean of the College of Science for providing this
opportunity to accomplish this research work.
Deepest gratitude to all consultants and other staff in the Department
of immunology at the Alhusein-Teaching Hospital for their assistance,
valuable advice, and consultation in choosing the subjects and clinical part of
the thesis.
I would like to express my profound thanks and sincere gratitude to my
husband for his encouragement and support that made it possible for me to
accomplish my study.
I would to express my thanks to all those who have contributed to the
completion of this work, all individuals who cooperated with me; and without
their help, this work would not have been accomplished. Finally, this work
could have not been accomplished without the cooperation of patients and
their families.
Alaa
Supervisor Declaration
We declare that this thesis was prepared under our supervision at the
Department of Biotechnology / College of Science / University of Baghdad,
in partial fulfillment of the requirement for the degree of Doctor of
Philosophy in Immunology/Biotechnology.
Signature:
Signature
Supervisor
Supervisor
Dr. Alice K. Melconian
Dr. Ali H. Ad’hiah
Professor
Professor
Department of Biotechnology
Tropical-Biological Research Unit
College of Science
College of Science
University of Baghdad
University of Baghdad
Date:
Date:
In view of the available recommendations, I forward this thesis for debate by
the examination committee.
Dr. Abdul kareem Al-kazaz
Assistant Professor
Head
Department of Biotechnology
College of Science
University of Baghdad
Committee Certification
We, the examining committee certify that we have read this thesis entitled "
Immunological and Biochemical profile of Alzheimer's Disease in a Sample
of Iraqi Patients", and have examined the Ph.D. student " Alaa Abd-Alhasan
Hamdan Al-Ganzawi" in its contents, and in our opinion it is accepted as a thesis
for the degree of Doctor of Philosophy in Biotechnology/ Immunology with the
average mark Excellent.
Chairman
Dr. Sabah N. Alwachi
Professor
15/ 11 / 2013
Member
Dr. Majid M. Mahmood
Professor
15/ 11 / 2013
Member
Dr. Amna N. Jasim
Assistant Professor
15/ 11/ 2013
Member
Dr. Hasan F. Al-Azzawi
Professor
15/11/ 2013
Member
Dr. Shahlaa M. Salh
Assistant Professor
15/ 11 / 2013
Supervisor
Dr. Alice K. Melconian
Professor
15/ 11 / 2013
Supervisor
Dr. Ali H. Ad'hiah
Professor
15/ 11 / 2013
Approved by the Dean of the College of Science, University of Baghdad
The Dean
Dr. Saleh M. Ali
Professor
15/ 11 / 2013
I
Summary
Summary
The present study is a trial to clear up the difficulties in diagnosis of
the Alzheimer's disease and to identify the high risk Alzheimer's disease
population.
The results presented in this study were based on analysis of data
from a total of 88 subjects: 30 Alzheimer's disease (AD), 28 vascular
dementia (VD), 10 Down's syndrome (DS), and 20 healthy controls (HC).
Based on information collected from the investigated subjects it was
possible to characterize them demographically in terms of age, duration of
disease, gender, educational status, family history of corresponding
illness, allergy to fish meat, as well as, cigarette smoking and alcohol
drinking.
These patients were collected from educational Alhussein hospital in
Karbala and hospice in governorates in Iraq (Karbala, qadesia, Alrashad
city in Baghdad, elderly house in Kademeia) during the period of October
2011 to September 2012. Several serological tests were performed to
detect C-reactive protein, total antioxidant capacity, Beta amyloid protein
, IL-1β, IL-17A,IL-10 Cytokines using enzyme linked immunosorbant
assay (ELISA); C3, C4, IgA, IgM, IgG, alpha 1-antitrypsin and lipid
profile.
Alzheimer's disease patients had the highest mean of age (76.9 ± 2.9
years) followed by VD patients (72.2 ± 1.7 years), also most cases of AD
patients (76.7%) had a duration of 6-15 years, while in VD, 89.3% of
patients had a duration of ≤ 5 years, both diseased groups showed a high
frequency of females than males (66.7 vs. 33.3% in AD and 57.1 vs.
42.9% in VD). Most of AD patients were illiterate (86.7%), while most
of VD patients had some sort of education (78.6%).
II
Summary
Six out of 30 AD patients (20.0%) were observed to have a family
history of the disease (father, mother or brother), while the corresponding
frequency in VD patients was higher (32.1%).
The highest level of Aβ was observed in AD patients (56.81 ± 4.19
pg/ml), followed by DS (34.20 ± 4.77 pg/ml) and VD (23.8 ± 1.64 pg/ml)
patients, while control were (9.87 ± 1.05 pg/ml), Distributing AD and VD
patients by gender revealed that females had a significantly higher serum
level of Aβ than males of both groups of patients (AD: 62.44 ± 5.5 vs.
46.57 ± 5.35 pg/ml; VD: 26.2 ± 2.3 vs. 20.6 ± 1.8 pg/ml), this protein
may play a pathogenic mechanism of AD. The highest serum level of
total cholesterol was observed in VD patients (264 ± 15 mg/dL), which
represented a significant (P ≤ 0.01) difference in comparison with AD,
while the serum level means of triglycerides in AD and VD patients were
(203 ±15 and 189 ± 11 mg/dL), respectively, which were not significantly
different, There was a significantly decreased serum level of HDL
cholesterol in AD and VD patients (33.4 ± 1.2, 41.5 ± 1.8 mg/dL,
respectively), The highest mean level of LDL cholesterol was observed in
VD patients (185.0 ± 15.2 mg/dL), and the difference was significant in
comparison with AD patients
(84.5 ± 7.7, 146.0 ± 10.0 mg/dL,
respectively), The mean serum level of VLDL cholesterols showed no
significant difference between AD and VD patients (40.7 ± 2.9 and 37.9 ±
2.1 mg/dL) respectively.
The lowest TAC was observed in AD patients (5.29 ± 0.46 nmol/μL)
as compared with VD patients (8.85 ± 0.40 nmol/μL), A common theme
between AD and VD patients was presented by a significant increased
serum level CRP (5.17 ± 0.52 and 4.39 ± 0.48 mg/dL, respectively). The
serum level of α1-antitrypsin was significantly increased in AD and DS
patients (275 ± 23 and 238 ± 10 mg/dL, respectively).
Summary
III
The result determining serum IgA level showed no significant difference
between VD patients (348 ± 35 mg/dL) patients and AD (397 ± 32
mg/dL), also There was no significant difference between the means of
IgG in AD and VD patients (1246 ± 118 and 996 ± 131 mg/dL,
respectively), there are approximated mean of serum IgM level, and there
was no significant difference between them.
Serum level of C3 was exceptionally and significantly increased in AD
patients (179 ± 10 mg/dL), as compared with VD patients (135 ± 9
mg/dL), The highest serum level of C4 was observed in AD patients (51.5
± 2.7 mg/dL), but the difference was not significant in VD patients (49.4
± 6.0 mg/dL).
No significant difference observed in serum level IL-1α between AD
and VD ( 3.79 ± 0.26, 3.25 ± 0.20 pg/ml) patients, the serum level of IL10 was approximated in VD and DS patients (3.39 ± 0.24, 2.77 ± 0.39
pg/ml, respectively), but was significantly(P≤ 0.05) increased in AD
patients (5.73±0.55pg/mL) as compared to the other group. The serum
level of IL-17A was significantly increased in AD and VD patients (6.28
± 0.35 and 5.32 ± 0.42 pg/ml) respectively. as compared with controls
(4.05 ± 0.28 pg/ml).
List of Contents
IV
List of Contents
Index
Title
Summary
List of contents
Pag
eI
IV
List of Tables
VII
List of Figures
IX
List of Abbreviations
X
Introduction
Introduction
Aims of Study
1
3
Chapter One : Review of Literature
1.1
1.2
Historical Background
Epidemiological Profile
4
5
1.3
Clinical Presentation
6
1.4
Pathogenesis
7
1.5
Aetiology and Risk Factors
9
1.5.1
Genetics
9
1.5.2
Lifestyle and Vascular Risk Factors
10
1.5.3
Inflammatory and Immunological Factors
13
1.5.4
Protective and Psychological Factors
16
1.6
Alzheimer’s Disease and Down’s Syndrome
17
1.7
Parameters of Present Study
18
1.7.1
Beta Amyloid
18
1.7.2
Lipid Profile
21
1.7.3
Total Antioxidant Capacity
22
1.7.4
C-reactive Protein
23
1.7.5
Alpha 1-antitrypsin
25
1.7.6
Immunoglobulins (IgA, IgG and IgM)
26
1.7.7
Complement Components C3 and C4
27
1.7.8
Cytokines
29
List of Contents
V
1.7.8.1
1.7.8.2
Interleukin-1α
Interleukin-10
30
32
1.7.8.3
Interleukin-17A
33
Chapter Tow: Subjects, Materials and Methods
2.1
2.2
Patients and Controls
Materials
35
36
2.2.1
Equipment, Plastic and Glassware
36
2.2.2
Laboratory Kits
36
2.3
Collection of Blood Samples
37
2.4
Laboratory Methods
37
2.4.1
Beta Amyloid1-40 (Aβ1-40) Assessment
37
2.4.2
Cholesterol Determination
39
2.4.3
Triglycerides Determination
40
2.4.4
High Density Lipoproteins (HDL) Cholesterol determination
40
2.4.5
Low Density Lipoproteins (LDL) Cholesterol determination
41
2.4.6
Very Low Density Lipoproteins (VLDL) Cholesterol
41
2.4.7
Total Antioxidant Capacity determination
41
2.4.8
High Sensitive C-reactive Protein (hsCRP) determination
43
2.4.9
Alpha1-antitrypsin, Immunoglobulins and Complement
45
2.4.10
Cytokines (IL-1α, IL-10 and IL-17A) determination
46
Statistical Methods
50
2.5
Chapter Three: Results and Discussion
3.1
3.1.1
Demographic Presentation of Study Groups
Age
52
52
3.1.2
Duration of disease
53
3.1.3
Gender
54
3.1.4
Educational Status
56
3.1.5
Family History
56
3.1.6
Allergy to Fish Meat
58
3.1.7
Cigarette Smoking
59
List of Contents
3.1.8
3.2
VI
Alcohol Drinking
Serum Level of Beta Amyloid (Aβ)
59
60
3.3
Lipid Profile
63
3.4
Total Antioxidant Capacity (TAC)
71
3.5
C-reactive Protein (CRP)
73
3.6
Alpha 1-antitrypsin (α1-antitrypsin)
75
3.7
Immunoglobulins A, G and M
77
3.8
Third and Fourth Components of Complement
79
3.9
Serum Level of IL-1α, IL-10 and IL-17A
81
3.9.1
Interleukin-1α
81
3.9.2
Interleukin-10
83
3.9.3
Interleukin-17A
85
3.10
Duration of AD and the Investigated Parameters
86
Conclusions and Recommendations
Conclusions
Recommendations
References
Appendix
Arabic Summary
92
93
94
List of Tables
VII
_________________________________________________________________________
List of Tables
Tables
Table 3-1
Table 3-2
Table 3-3
Table 3-4
Table 3-5
Table 3-6
Table 3-7
Table 3-8
Table 3-9
Table 3-10
Table 3-11
Table 3-12
Table 3-13
Table 3-14
Table 3-15
Table 3-16
Titles
Age distribution in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Alzheimer's and vascular dementia patients distributed
by duration of disease.
Alzheimer's and vascular dementia patients distributed
by gender.
Alzheimer's and vascular dementia patients distributed
by educational status.
Alzheimer's and vascular dementia patients distributed
by family history.
Alzheimer's and vascular dementia patients distributed
by allergy to fish meat.
Alzheimer's and vascular dementia patients distributed
by cigarette smoking.
Alzheimer's and vascular dementia patients distributed
by alcohol driniking.
Serum level of beta amyloid in Alzheimer's, vascular
dementia and Down's syndrome patients and controls.
Serum level of beta amyloid in Alzheimer's and
vascular dementia distributed by gender.
Serum level of total cholesterol in Alzheimer's,
vascular dementia and Down's syndrome patients and
controls.
Serum level of triglycerides in Alzheimer's, vascular
dementia and Down's syndrome patients and controls.
Serum level of high density lipoproteins cholesterol in
Alzheimer's, vascular dementia and Down's syndrome
patients and controls.
Serum level of low density lipoproteins cholesterol in
Alzheimer's, vascular dementia and Down's syndrome
patients and controls.
Serum level of very low density lipoproteins
cholesterol in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Serum level of total antioxidant capacity in
Alzheimer's, vascular dementia and Down's syndrome
patients and controls.
Page
51
52
53
55
56
57
58
59
59
60
62
63
63
64
64
71
List of Tables
VIII
_________________________________________________________________________
Table 3-17
Table 3-18
Table 3-19
Table 3-20
Table 3-21
Table 3-22
Table 3-23
Table 3-24
Table 3-25
Table 3-26
Table 3-27
Serum level of C-reactive protein in Alzheimer's,
vascular
dementia and Down's syndrome patients and controls.
Serum level of alpha 1-antitrypsin in Alzheimer's,
vascular dementia and Down's syndrome patients and
controls.
Serum level of IgA in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of IgG in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of IgM in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of C3 in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of C4 in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of IL-1α in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of IL-10 in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Serum level of IL-17A in Alzheimer's, vascular
dementia and Down's syndrome patients and controls.
Means of investigated parameters distributed by
duration of disease in Alzheimer's patients.
73
75
76
76
77
78
79
80
82
84
88
List of Figures.
...........
..............................................................
List of Figures.
Figures
Figure 1-1
Figure 1-2
Figure 1-3
Figure 2-1
Figure 2-2
Figure 2-3
Figure 2-4
Figure 2-5
Figure 2-6
Figure 3-1
Figure 3-2
Figure 3-3
Figure 3-4
Figure 3-5
. ..
.
Titles
Page
. . . .. .. .... ... .... . . .. .. ..
.
.. . . . . .... .. . .. .. . ... ... .. ... .... .. ... . . . .. . . ... ... . . ..
. . . ... .. .
immune responses in Alzheimer’s disease patients
.. .. . .. .. . .. .. .β (Aβ) levels in sporadic Alzheimer’s ..
. .. ... . . .. . .. .. . . .. . ... .. . .. . ... . ... . . .. . .. ... .. . . . ..
... . . ..... .. . .. ... . . ... .
.. .. . ... ..... ...... .. .... . .. .. .
..
.. .. . ... ..... ...... . ... .... .. . .. .. ..... ... .. .
..
.. .. . ... ..... ...... .. . .... . .... ... ...... .. ..... ... . .
..
.. .. . ... ..... ....... .1α.
..
.. .. . ... ..... ....... ....
..
.. .. . ... ..... ....... .. . . .
..
. . ... . .. . ... .... .. . ..... ... .. . .. .. .. .. ... . . . . . . .. ... . ..
. ... . . ... .. . .. . . ... .. . .... .. . .. .. .... . .. .. .... .. . .
. . . . ..... . . .. . .... .... .. .... ... . ... .. .
. .. . .. .... . .. . . ..... ... .. . .. .. .. .. ... . . . . . . .. ... . . ... . . ..
... .. . .. . . ... .. . .... .. . .. .. .... . .. .. .... .. . . . . . . ...
. . . . .. . .... .... .. .... .... ... .. .
. .. . . . .. . ... . ... . . .. ... . . . .. . ... .... .. . ..... ... .. . .. .. .. ..
.. ... . . . . . . .. ... . . ... . . .. . .. . .. . . ... .. . .... .. . .. .. ....
. .. .. ...... . .. . . . ..... . . .. . .... .... .. .... .... ... ..
. . . . . .. . ... . ... . . .. ... . . . .. . ... .... .. . ..... ... .. . .. .. .. ..
.. ... . . . . . . .. ... . . ... . . ... .. . .. . . ... .. . .... .. . .. .. ....
. .. .. ...... . .. . . . ..... . . .. . .... .... .. .... .... ... .. .
. ... ... . .. .. . ... .... . . .. ... . . ... . ... .... ... ..... ... .. . .. ..
.. .. .. ... .. . . .. .. ... .. ... . .... .. ... .. ... .. . .... ... .. .. ....
. .. .. ...... . .. . . . ..... . . .. . .... .... .. .... .... ... .. .
.
.
List of Abbreviations
X
___________________________________________________________________________
List of Abbreviations
AD
ADRDA
Alzheimer's disease
Alzheimer's Disease and Related Disorders Association
ApoA
Apolipoprotein A
APOE
apolipoproteinE
APP
apolipoprotein
BBB
blood–brain-barrier
BSA
bovine serum albumin
C3
Third component of complement
C4
fourth component of complement
CD
Cluster of Differentiation
CNS
Central nervous system
COX-2
Cyclooxygenase type 2
CSF
cerebrospinal fluid
CSFs
Colony timulating factors
CTLA8
T lymphocyte-associated antigen 8
DS
Down syndrome
DW
Distilled water
ELISA
Enzyme-linked immunosorbent assay
EDTA
Ethylene diamine tetra –acetic acid
fAβ
Aβ fibrils
HC
HDL
healthy controls
High density lipid
HRP
anti-rabbit antibody
hsC-RP
High sensitive C-reactive protein
ICAM-1
intercellular adhesion molecule-1
IFN- γ
Interferon-gamma
IG
Immunoglobulin
IgA
Immunoglobulin A
List of Abbreviations
XI
___________________________________________________________________________
IgG
Immunoglobulin G
IgM
Immunoglobulin M
IL-10
Interleukin ten
IL-17A
Interleukin 17 A
IL-1ra
IL-1 receptor antagonist
IL-1α
Interleukin alpha one
iNOS
inducible nitric oxide synthase
kDa
Kilodalton
LDL
Low density lipid
MHC
Major histocompatiblity complement
MMP
matrix metalloproteinase
MW
Molecular weight
NINCDS
National Institute of Neurological and Communicative Disorders
PBMCs
peripheral blood mononuclear cells
PBS
phosphate buffer saline
PET
positron emission tomography
PGE2
prostaglandin-E2
RNS
reactive nitrogen species
ROS
reactive oxygen species
S.E.
Standard error
SC
secretory component
TAC
Total antioxidant capacity
TMB
Tetramethylbenzidine
TNF- α
Tumor necrosis factor-alpha
VCAM- 1
vascular-cell adhesion molecule- 1
VD
Vascular dementia
VLDL
Very low density lipid
α-1ntitrypsin
Alpha 1-Antitrypsin
βA
Βeta-amyloid
Introduction
۱
================================================================
Introduction
Alzheimer's
disease
(AD)
is
an
age-related
heterogeneous
neurodegenerative disorder associated with progressive functional decline,
dementia and neuronal loss, and it is considered as a major public health
problem with a huge associated impact on individuals, families, healthcare
system and society (Selkoe, 2002).This incurable and degenerative disease is
usually diagnosed in people over 65 years of age, although a less-prevalent
early-onset AD can occur much earlier (Qiu et al., 2009). In Western
societies, AD accounts for the majority of clinical senile dementia and by
2050 the number of patients with AD is expected to rise from 4.6 to 16
million cases in the USA, while worldwide statistical projections predict
more that 45 million of AD patients within the above year, and further
epidemiological estimations suggested a number of 100 million (Alzheimer’s
Association, 2010).
The major pathological hallmarks of AD include presence of abnormal
proteinaceous deposits known as senile plaques and neuroifbrillary tangles�
(NFTs), along with extensive neuronal loss in speciifc cortical an��
subcortical regions such as the nucleus basalis of Meynert and the
hippocampus. Senile plaques are composed primarily of the protein fragment
�-amyloid (A�), and are generally thought to be formed extracellularly,
although there is also evidence from murine models which suggests that the
process of oligomerization and subsequent deposition begins in intracellular
compartments (Tang, 2009). However, aetiologically, AD is a multifactorial
disease, in which older age is the strongest risk factor, suggesting that the
aging-related biological processes may be implicated in the pathogenesis of
the disease. Furthermore, the strong association of AD with increasing age
may partially reflect the cumulative effect of different risk and factors over
the lifespan, including the effect of complex interactions of genetic
Introduction
۲
================================================================
susceptibility, psychosocial factors, biological factors (vascular and
immunological), and environmental exposures experienced over the lifespan
(Fratiglioni et al., 2008).
The concern of present study is vascular and immunological factors,
which can collectively be assigned as biomarkers of AD in blood of patients.
In the last years many efforts were done to find disease specific and reliable
blood biomarkers, and accordingly different candidates such as α1antitrypsin, complement factor H, α-2-macroglobulin, apolipoprotein J
(ApoJ) and ApoA-1 have been proposed. In 2007, with a combined
multivariate analysis of 18 plasma signaling and inflammatory proteins (for
instance, IL-1α, IL-3, TNF-α), Ray and colleagues identified a profile that
might be indicative of AD (Ray et al., 2007). The role of inflammation with
microglia activation has also been believed to play a role in AD
pathogenesis, but the presence of inflammatory markers in serum or plasma
has not been clear, and inflammatory molecules, such as IL-1β, TNF-α, IL-6,
C-reactive protein (CRP) and α1-antichymotrypsin showed contrasting
results (Teunissen and Scheltens, 2007). Furthermore, Tan et al. (2007)
observed that high levels of peripheral blood mononuclear cell (PBMC) of
the inflammatory cytokines, such as IL-1 or TNF-α, are associated with an
increased risk of developing AD. Accordingly, it has become increasingly
clear that immunological processes play a significant role in the
pathophysiology of AD, and neuro-inlfammation is characterize� by the
activation of astrocytes and microglia and the release of pro-inflammatory
cytokines and chemokines (Broussard et al., 2012). In addition to these
immunological markers, evidence has been accumulated that cholesterol
metabolism plays a role in AD, and total serum cholesterol may be a marker
of the disease, because high level of serum cholesterol was associated with
an increased risk of incident AD (Anstey et al., 2008; Maulik et al., 2013).
Introduction
۳
================================================================
Aims of Study
The presented introductory theme promoted the present study to be
carried out with the aims to evaluate some vascular and immunological
parameters that may have impact on the aetiopathogenesis of AD in a sample
of Iraqi patients, and to reach a better understanding of these profiles, two
related groups of patients were also investigated; they were vascular (VD)
dementia patients and children with Down's syndrome (DS). Such scopes
were targeted through the assessment of the following parameters in sera of
the investigated groups:
•
Beta amyloid1-40.
•
Lipid profile.
•
Total antioxidant capacity.
•
C-reactive protein.
•
α1-antitrypsin.
•
Immunoglobulins (IgA, IgG and IgM).
•
Complement components C3 and C4.
•
IL-1α, IL-10 and IL-17A.
Chapter One: Review of Literature
4
===================================================================
.. .....
....
.
... .. . ... ... ..... . ..
.
Alzheimer’s disease is the most common form of dementia. It is a
degenerative and incurable disease and affects most (up to 75%) of the more
than 35 million people suffering from dementia worldwide, and prevalence is
believed to double every 20 years. There are two main forms of the disease;
familial and sporadic AD. The former affects people younger than 65 years old,
while the latter occurs in adults aged 65 years and older (Qiu et al., 2009). The
disease has a major impact not only on the sufferers but also on persons caring
for them, as well as the entire society. The aetiological factors are mostly
unknown, but there is increasing evidence that certain risk factors are engaged
in the development of the disease, such as genetic, immunological and vascular
factors. The increasing prevalence of AD is also attributed to population aging,
which is almost seen worldwide (Povova et al., 2012).
.. .. . ...... .. ... .........
.
Alzheimer’s disease was discovered in 1906 by the German neurologist
and psychiatrist Dr. Alois Alzheimer. The disease was initially observed in a
51-year-old woman named Auguste. Her family brought her to Dr. Alzheimer
in 1901 after noticing changes in her personality and behavior. The family
reported
problems
with
memory,
difficulty
speaking
and
impaired
comprehension. Dr. Alzheimer later described Auguste as having an aggressive
form of dementia, manifesting in memory, language and behavioral deficits.
After following-up her care for five years, he noted many abnormal symptoms,
including difficulty with speech, agitation, and confusion. Following her death
in 1906, Dr. Alzheimer performed an autopsy, during which he found dramatic
Chapter One: Review of Literature
5
===================================================================
shrinkage of the cerebral cortex, fatty deposits in blood vessels and atrophied
brain cells. He also discovered NFTs and senile plaques, which have become
indicative of AD. The condition was first discussed in medical literature in 1907
and named as AD after Alzheimer in 1910 (Reviewed by Bethune, 2010).
.. .. .. .....
... ... .. ... .. ... .
Pooled data of population-based studies in Europe suggested that the agestandardized prevalence in people 65+ years old is 6.4% for dementia and 4.4%
for AD (Lobo et al., 2000). In the USA, the study of a national representative
sample of people aged greater than 70 years yielded prevalence for AD of 9.7%
(Plassman et al., 2007). Worldwide, the global prevalence of dementia was
estimated to be 3.9% in people aged 60+ years, with the regional prevalence
being 1.6% in Africa, 4.0% in China and Western Pacific regions, 4.6% in Latin
America, 5.4% in Western Europe, and 6.4% in North America (Ferri et al.,
2005).
More than 25 million people in the world are currently affected by
dementia, most suffering from AD, with around 5 million new cases occurring
every year (Brookmeyer et al., 2007). Among developed nations, approximately
1 in 10 older people (65+ years) is affected by some degree of dementia,
whereas more than one third of very old people (85+ years) may have dementiarelated symptoms and signs (Corrada et al., 2008). In low- and middle-income
countries, it has been estimated that the overall prevalence of AD in developing
countries was 3.4% (Kalaria et al., 2008), while the Dementia Research Group
found that the prevalence of dementia in people aged 65+ years in seven
developing nations varied widely from less than 0.5% to more than 6%, which
is substantially lower than in developed countries, and furthermore, the
Chapter One: Review of Literature
6
===================================================================
prevalence rates of dementia in India and rural Latin America were
approximately a quarter of the rates in European countries (Llibre et al., 2008).
.. .. ..... ... .... .........
.
The typical clinical presentation of AD that of insidious progressive
impairment of episodic memory representing early involvement of medial
temporal lobe structures with the emergence of additional deficits such as
aphasia, apraxia, agnosia, and executive deficits as the disease progresses.
Findings from longitudinal studies indicate that neuropsychological deficits in
multiple cognitive domains are evident several years in advance of a diagnosis
of AD (Blennow et al., 2006). A recent meta-analysis reported that the largest
deficits in preclinical AD exist in the domains of perceptual speed, executive
functioning, and episodic memory with smaller deficits in the domains of verbal
ability, visuospatial skills, and attention. This is characterized clinically by
initial forgetfulness for daily events with progressive involvement of language
skills, decision making, judgment, orientation, recognition, and motor skills
(Gallagher et al., 2010). Neuropsychiatric symptoms are frequently observed
and occur in 60–98% of patients with dementia. They are a significant source of
distress for patients and families and a major determinant of outcomes such as
length of hospital stay and nursing home placement. They ordinarily increase
with increasing disease severity but are observed early in the disease process
and have been documented in 30–75% of patients with mild cognitive
impairment. Apathy, anxiety, depression, and agitation occur most frequently.
Delusions are also common and include themes of theft, intruders, imposters, or
other ideas of persecution, reference, or infidelity. Visual and auditory
hallucinations are the most common perceptual abnormalities although somatic,
Chapter One: Review of Literature
7
===================================================================
olfactory and tactile hallucinations have also been reported (Gallagher et al.,
2011).
.. .. ...........
..
The two core pathological hallmarks of AD are amyloid plaques and
NFTs. It has been suggested that a deposition of Aβ triggers neuronal
dysfunction and death in the brain, and this neuronal dysfunction and death was
thought to be due to a toxic effect of the total amyloid load (Figure 1-1).
Furthermore, specific alterations in Aβ processing have also been demonstrated,
such as the cleavage of amyloid precursor protein (APP) into Aβ peptides (Aβ1–
40
and Aβ1–42). The Aβ1–42 peptide aggregates more readily than Aβ1–40, and the
ratio of these two isoforms is influenced by the pattern of cleavage from APP
by α, β, and γ secretases (Hardy, 2006), and small oligomers of Aβ can be more
toxic than mature fibrils. In this regard, Aβ56 is suggested to be a peptide of
particular interest because it has been shown to be negatively associated with
cognitive decline in an APP mouse model and induces memory deficits when
injected into rat brain (Morris and Mucke, 2006). In addition, increases in Aβ
might result from neuronal damage caused by another process, and Aβ
sequence, Aβ concentration, and conditions that destabilise Aβ are thought to be
important factors (Nerelius et al., 2010).
The pathogenesis of AD can also be discussed in the ground of a
microtubule-associated protein, which is known as tau. Tau is a major
constituent of NFTs and changes in it and consequent NFT formation has been
observed to be triggered by toxic concentrations of Aβ, but pathways linking
Aβ and tau are not clearly understood, although several hypotheses have been
proposed (Small and Duff, 2008). Tau is a soluble protein, but insoluble
aggregates are produced during the formation of NFTs, which disrupt the
Chapter One: Review of Literature
8
===================================================================
structure and function of the neuron. Tau monomers first bind together to form
oligomers, which then aggregate into a β sheet before forming NFTs (MerazRíos et al., 2010). Once filamentous tau has formed, it can be transmitted to
other brain regions, and it has been demonstrated that an injection of mutant
pathological tau can induce the formation of tau filaments in wild-type mice
(Clavaguera et al., 2009). However, post-mortem measurement of each of these
classic pathological hallmarks only explains to a limited extent the expression
of dementia in the population (Matthews et al., 2009), and numerous other
potentially modifiable factors (i.e. risk factors) also contribute to the clinical
presentation of dementia in AD (Ballard et al., 2011).
.. . .. .. . .. : Amyloid cascade hypothesis: Amyloid precursor protein (APP) is
processed into amyloid β (Aβ), which accumulates inside neuronal cells and
extracellularly, where it aggregates into plaques. In the amyloid cascade
hypothesis, these Aβ deposits are toxic and cause synaptic dysfunction and
neuronal cell death (Ballard et al., 2011).
Chapter One: Review of Literature
9
===================================================================
.. .. .... . ... . .. .. ..... .... ... .. .
Based
on
epidemiological
studies,
neuroimaging
methods
and
neuropathology research, three aetiological hypotheses (genetic, lifestyle,
vascular, and psychosocial) of AD development have been discussed in the
literature, and further discussions have also introduced the inflammatory and
immunological hypotheses.
. .... .. ... .... .
Genetically, AD is a heterogeneous disorder with both familial and
sporadic forms. Early-onset familial AD is often caused by autosomal dominant
mutations (for instance, mutations in APP, presenilin-1, and presenilin-2 genes),
but they account for only 2-5% of all Alzheimer patients (Blennow et al., 2006).
However, the majority of AD cases are sporadic and present considerable
heterogeneity in terms of risk factor profiles and neuropathological features.
First-degree relatives of Alzheimer patients have a higher lifetime risk of
developing AD than the general population or relatives of non-demented
individuals (Green et al., 2002). In addition, some studies suggest that the
familial aggregation of AD can only be partially explained by known genetic
components such as the apolipoprotein E (AOOE) 44 allele, indicating that other
susceptibility genes may be involved (Huang et al., 2004). The AOOE 44 allele
is the only established genetic factor for both early- and late-onset AD;
therefore it is considered as a susceptibility gene for AD, but it is also suggested
that it is neither necessary nor sufficient for AD development, although
increasing number of the AOOE ε4 alleles, the risk of AD increases and the age
of AD onset decreases, in a dose-dependent manner (Qiu et al., 2004). The risk
effect of AOOE 44 allele on AD decreases with increasing age, and overall
approximately 15-20% of Alzheimer cases are attributable to the 44 allele
Chapter One: Review of Literature
10
===================================================================
(Bertram et al., 2007). Other candidate genes with polymorphisms affecting the
risk of the development of AD have been studied. Predominantly, these are
secretase genes (beta-site amyloid precursor protein-cleaving enzyme 1),
presenilin 1, peptidase genes (endothelin-converting and insulin-degrading
enzymes), microtubule and cytoskeletal genes (microtubule-associated protein
tau), synaptic genes (ATP-binding cassette A1 transporter), anti-apoptotic genes
(IL-1), protease genes (angiotensin-converting enzyme) and other genes such as
the gene for APP, but with inconsistent findings (Bettens et al., 2013).
.. . ..........
..... .....
.... ..... .. .......
.
Although several AD risk factors are genetic in nature, others are
determined by environmental or lifestyle influences and may be amenable to
modification, and have been suggested to be associated with a higher risk of
dementia including AD. They include smoking and alcoholism, obesity and
high total cholesterol levels, together with vascular morbidity, such as
hypertension, diabetes mellitus and asymptomatic cerebral infarction, and these
factors are also subjected to the aging-related biological processes that are may
be implicated in AD (Povova et al., 2012).
Earlier cross-sectional studies often reported a lower prevalence of AD
among smokers compared with non-smokers (Fratiglioni and Wang, 2000).
This seemingly protective effect was probably due to survivor bias since the
proportion of smokers among the prevalent cases was smaller, and when
incident cases of AD were studied, however, the situation was completely
reversed (Hill et al., 2003). That is, numerous analytical studies found a
significantly increased risk of AD associated with cigarette smoking, especially
in apoE4 allele non-carriers (Tyas et al., 2003; Aggarwal et al., 2006). Metaanalyses of these analytical studies concluded that smoking was associated with
Chapter One: Review of Literature
11
===================================================================
an increased risk of the development of AD (Anstey et al., 2007; Peters et al.,
2008). In addition to smoking, it is also well-recognized that alcohol abuse can
cause alcohol dementia. Moreover, middle-aged heavy drinkers, especially
apoE4 allele carriers, were found to have a more than 3-fold higher risk of
dementia and AD later in their lives (Anttila et al., 2004). On the other hand,
the risk of developing dementia and AD was reduced in light and moderate
alcohol consumers. In heavy consumers, alcohol clearly damages the brain, and
even light to moderate alcohol consumption was found to be related to brain
atrophy (Ding et al., 2004; Paul et al., 2008).
With respect to vascular factors, longitudinal studies such as the
Cardiovascular Risk Factors, Aging, and Dementia (CAIDE) study have found
midlife hypertension, hypercholesterolemia and obesity to be associated with
increased risk of dementia and AD in later life (Lindsay et al., 2002). Further
analyses revealed that a clustering of risk factors was observed to increase AD
risk in an additive fashion. A dementia risk score using data gathered during the
CAIDE study predicted dementia with a sensitivity of 0.77, specificity of 0.63,
and negative predictive value of 0.98 over 20 years of follow up. This score
included variables such as age (≥ 47 years), low education, hypertension,
hypercholesterolemia, and obesity (Gallagher et al., 2011).
There is also a great deal of interest in developing approaches to help
reduce the risk of AD in later life through identifying individuals who might
benefit from intensive lifestyle consultations and pharmacological interventions
in earlier life. In accordance with such theme, a recent systematic review
concluded that the evidence for single clinically defined vascular risk factors
was inconsistent at best while the strength of the association was increased by
identifying interactions between risk factors such as hypertension and diabetes
(Gallagher et al., 2010), and furthermore, cerebrovascular diseases such as
Chapter One: Review of Literature
12
===================================================================
stroke may increase the risk of cognitive impairment and dementia, and may
contribute to the progression of AD (Kim et al., 2011).
There is a good evidence that a first-ever stroke increases the risk of
cognitive impairment and, furthermore, that recurrent strokes promote the
appearance of dementia of a progressive-type fitting criteria for AD (Srikanth et
al., 2004). It is also recognized that dementia in older age occurs due to a
combination of cerebrovascular disease and AD, but the latter being
characterized by increased formation of beta amyloid plaques and NFTs in the
brain (Srikanth et al., 2006). Stroke is associated with a state of acute focal
reduction in cerebral blood flow (i.e. cerebral hypoperfusion) in a defined
vascular territory and consequent oxidative stress (Moskowitz et al., 2010).
Such a state of oxidative stress may promote regional neuronal death but may
also promote the occurrence of pathological changes of AD (Iadecola, 2010).
Apart from clinical stroke itself, vascular risk factors (such as hypertension,
diabetes mellitus, and obesity) that develop from early to mid-life have been
shown to be associated with the later appearance of cognitive impairment and
dementia. Over the lifespan, there may be interplay between cerebrovascular
disease or its risk factors that may initiate or promote the expression of clinical
AD in later life (Gorelick et al., 2011).
The vascular mechanisms underlying these associations are poorly
understood, with arteriosclerosis, hypertensive angiopathy, and microvascular
disease being potential pathways, each of which involves vascular oxidative
stress. It has been suggested that such disease processes may lead to chronic
hypoperfusion of structures such as the deep white matter, and more strategic
areas such as the hippocampus (Drummond et al., 2011).
.
Chapter One: Review of Literature
13
===================================================================
.. . ........
. . . .. .. ......
..
... .. . ... ... . ...... .
In addition to Aβ and tau protein aggregates, the presence of immunerelated antigens and cells around amyloid plaques in the brains of patients with
AD has been reported since the 1980s (Rogers et al., 1988). In the 1990s,
additional findings of activated complement factors, cytokines and a wide range
of related receptors in the brain of AD patients led to the concept of
neuroinflammation, which suggests that immunological processes in the brain
are likely to be involved in the pathology of degenerative diseases of the central
nervous system (CNS) (Bales et al., 2000).
It has been almost agreed that brain inflammation is the pathological
hallmark of AD, and it clearly occurs in pathologically susceptible regions of
brain in AD patients, with increased expression of acute-phase proteins and proinflammatory cytokines (Di Bona et al., 2008; Di Bona et al., 2009; RubioPerez et al., 2012). The cells responsible for the inflammatory reaction are
microglia, astrocytes, and neurons. These activated cells have been shown to
produce high levels of inflammatory mediators such as pro-inflammatory
cytokines and chemokines, prostaglandins, leukotrienes, thromboxanes,
coagulation factors, free radicals as reactive oxygen species and nitric oxide,
complement factors, proteases and protease inhibitors, and C-reactive protein
(CRP) (Finch and Morgan, 2007). Such findings support the hypothesis that Aβ
plaques and tangles stimulate a chronic inflammatory reaction, and
inflammatory mediators, in turn, enhance APP production and the
amyloidogenic processing of APP to induce Aβ4-2 peptide production. These
circumstances were also found to inhibit the generation of a soluble APP
fraction, which has a neuroprotective effect (Lindberg et al., 2005). On the
contrary, Aβ induces the expression of pro-inflammatory cytokines in glial cells
in a vicious cycle (Pellicanό et al., 2010).
Chapter One: Review of Literature
14
===================================================================
To date, the timing with which neuroinflammation is believed to influence
AD is unknown; however, clinical and experimental evidence from different
transgenic models has suggested that a pro-inflammatory process might precede
plaque deposition (Ferretti and Cuello, 2011), and a paper has correlated the
increased levels of CRP with the formation of senile plaques (Strang et al.,
2012). C-reactive protein has been shown to exist in two forms: the monomeric
form, which has pro-inflammatory properties; and the circulating pentamer
form. It has been demonstrated that the aggregated forms of Aβ plaques lead to
the formation of the pro-inflammatory monomeric form of CRP, which
exacerbates local inflammation (Eisenhardt et al., 2009).
There is currently much evidence suggesting the involvement of a
systemic immune response in AD, and numerous investigations suggest that in
addition to the CNS cells, blood-derived cells can also be responsible for the
inflammatory response and seem to accumulate in the AD brain (Bonotis et al.,
2008; Miscia et al., 2009; Liu et al., 2010). It has been demonstrated that
neuroinflammation is able to induce the efflux of proteins, such as Aβ, or
inflammatory mediators from CNS across the blood–brain-barrier (BBB) and
this may cause systemic immune reaction and recruitment of myeloid or
lymphocytic cells into the CNS. In this regard, it is known that BBB exerts a
“monitoring role” between the immune system and AD to protect the brain from
the entry of macromolecules, like immunoglobulins, and cells, including
immunocompetent cells (Pellicanό et al., 2012). Furthermore, a recent
assumption supposes that microvascular diseases, often associated with AD,
microtraumas and inflammation could cause the abnormal permeability of the
BBB. The consequence of this impairment is the anomalous presence of serum
proteins in the cerebrospinal fluid and in the brain, including Aβ. In the brain
Chapter One: Review of Literature
15
===================================================================
Aβ can bind astrocytes, starting a degenerative and inflammatory process (Sardi
et al., 2011).
Under physiological conditions T lymphocytes are few in the brain,
although they are able to cross the BBB. It has been demonstrated that Tlymphocyte number increases in AD patients, especially in the hippocampus
and temporal cortex. Herein, activated microglial cells have been observed with
an increase in the expression of major histocompatibility complex (MHC)
molecules of the classes I and II, and consequently allow the migration of T
cells. By this pathway, communication between the CNS and the immune
system in AD could influence both the lymphocyte distribution in the blood and
the production of immune mediators (Britschgi and Wyss-Coray, 2007).
Therefore, despite T cells being able to enter the brain tissue, it is also possible
that T cells exert their effects without entering the CNS, and in agreement with
such theme, peripheral blood mononuclear cells (PBMCs) from AD patients
have been demonstrated to produce higher levels of pro-inflammatory
cytokines, such as IL-1β and LL-6, as compared with PBMCs from control
subjects (Di Bona et al., 2008; Di Bona et al., 2009). It has also been shown
that Aβ stimulates macrophage inflammatory protein (MPP)-1α overexpression
by peripheral T cells and its receptor CCR5 expression on brain endothelial
cells, and this is necessary for T cells to cross the BBB (Man et al., 2007).
Moreover, other altered immune parameters were documented, such as
decreased percentages of naive T cells and an increase of memory T cells, an
increased number of CD4+ T lymphocytes that lack the co-stimulatory
molecule CD28, and a reduction of CD4+CD25 high regulatory T cells (Larbi
et al., 2009). A hypothesis that supports the involvement of immune system in
the pathogenesis of AD is presented in figure (1-2).
Chapter One: Review of Literature
16
===================================================================
. .. .. .. . .. : Communication between the central nervous system and systemic
immune responses in Alzheimer’s disease patients (Martorana et al., 2012).
.. . .........
...... .... .. ... .. . ... ... . .....
Factors that have been reported to be protective from population studies
include regular fish consumption, moderate wine intake, and higher educational
status (Solfrizzi et al., 2011). There is also now a significant amount of
epidemiological data, which suggests that individuals who are more socially
and physically active and engage in more cognitively stimulating activities are
at decreased risk of developing dementia and AD (Middleton and Yaffe, 2009).
A number of psychological factors have also been found to be important.
Depressive symptoms frequently precede the onset of cognitive decline by a
short interval but depression occurring many years (>25 years) in advance of
Chapter One: Review of Literature
17
===================================================================
AD has been reported to be a risk factor. Psychological distress and loneliness
have been reported to increase risk and investigators have postulated that stress
effects may be mediated by the toxic impact of glucocorticoids and
neuroendocrine dysregulation upon the hippocampus and limbic structures.
Having a greater sense of purpose in life and conscientiousness appear to be
protective and have both been independently associated with reduced risk of
AD (Vilalta-Franch et al., 2012).
.. .. Alzheiee r’e Dieeaee aed Dwws’s Syndrmme.
Down’s syndrome (D,), which is almost always caused by the presence of
three complete copies of chromosome 21 secondary to meiotic non-disjunction,
is the most common chromosomal disorder and is also the most common
genetic cause of cognitive impairment. Among its consequences, by the age of
30, individuals with DS invariably develop amyloid plaques and NFTs and,
beginning in their 40s and continuing through their 70s, up to 75% of people
with DS develop dementia (Zigman et al,, 1996). Alzheimer’s disease in DS is
linked to the presence of three copies of the APP gene, which resides on
chromosome 21. This gene leads to increased APP mRNA and protein
expression, as well as higher levels of Aβ, and elderly adults with Dw who had a
microdeletion resulting in APP disomy did not develop dementia or classic AD
neuropathology (Zigman, and Lott, 2007).
The idea that the dementia associated with DS is in fact AD is supported
on many levels. Genetically, the root cause is the increased flux of wild-type
APP, consistent with the amyloid hypothesis. In vitro, DS cells recapitulate the
same stages of dysfunction; particularly of the endocytic pathways, as cells
from non-DS individuals with AD (Shi , 2012). Changes in blood biomarkers,
as well as the little information that is available with regard to cerebrospinal
Chapter One: Review of Literature
18
===================================================================
fluid (CSF) markers in patients with DS, are consistent with changes in the
blood and CSF in the non-DS AD population. Results of amyloid positron
emission tomography (PET) imaging from individuals with DS are consistent
with those from non-DS individuals with AD. The amyloid plaques and tau
tangles found in individuals with DS at autopsy are identical to those found in
the general AD population, and their location and progression mirrors that
observed in non-trisomic adults with AD (Ness et al., 2012). Moreover, the
apolipoprotein E (ε4) genotype is associated with a higher risk of AD and an
earlier onset of dementia in people with DS, in the same way as in the general
population. Finally, similarly to patients with AD, after decades of stable
cognitive and functional performance in DS, there is a progressive cognitive
decline leading to total dependency and death (Wilcock, 2012).
.
.. .. .. ...
.. .. .. ... ............
..
The present study dealt with vascular, immunological and inflammatory
serum parameters (beta amyloid, lipid profile, total antioxidant capacity, CRP,
α1-antitrypsin, Immunoglobulins [IgA, IgG and IgM], complement components
C3 and C4 and cytokines [IL-1α, LL-10 and IL-17A]) that can aid in the
understanding of these profiles in AD. It is worth to mention and for the best
knowledge of the investigator, none of these evaluations has been presented for
AD in Iraqi patients.
. .... ..... .. . . .. ..
Beta Amyloid (Aβ) is a 38-43 kDa peptide derived from the proteolytic
cleavage of its parent molecule, the A,,,
and Aβ forms the core of the
characteristic deposits observed in the AD brain, namely senile plaques and
cerebrovascular amyloid angiopathy (Masters et al., 1985). In the human brain,
Chapter One: Review of Literature
19
===================================================================
there are two main forms of Aβ. Under normal physiological conditions, the
predominant Aβ species is 40 amino acids long (Aβ1–40) (Kuo et al., 1996). In
AD, there is an accumulation of Aβ1–42, the longer form of Aβ, and it is thought
that Aβ1–42 is a more toxic species as it aggregates much more readily than Aβ1–
40
and thus may provide the seed for further Aβ deposition and related
pathologies (Wirths et al., 2004).
Although Aβ is produced by almost all cells in the body, a physiological
function for the peptide has not been determined. The ‘amyloid hypothesis’
posits that Aβ is central to the pathogenesis of AD (as reviewed early in this
chapter). However, researchers are still grappling with a number of fundamental
questions relating to how this small peptide leads to the formation of plaques
and how the pathogenic cascade of events contributes to neurodegeneration and
dementia. Moreover, the significance of Aβ in the peripheral blood is a matter
of controversy in AD, especially when plasma Aβ is employed as a diagnostic
marker (Bates et al., 2009).
Investigations into Aβ1–40 and Aβ1–42 levels in plasma have yielded
contradictory results, making interpretation difficult and severely limiting the
diagnostic utility of plasma Aβ measurement. As can be seen in figure 1-3,
cross-sectional studies have not revealed an association between dementia
severity and plasma Aβ levels and there is a high degree of variability between
reported findings, and there is significant overlap between plasma Aβ1–40 and
Aβ1–42 levels between control and AD subjects. It has also been demonstrated
that long-term use of medications may influence plasma Aβ1–42 levels (Forsberg
et al., 2008); a matter that add a further complication.
Few longitudinal studies have been conducted to determine whether
baseline differences in plasma Aβ levels can be detected. Again, the results
from these studies are contradictory. Higher baseline levels of Aβ1–42, but not
Chapter One: Review of Literature
20
===================================================================
Aβ1–40 have been associated with increased risk of AD over a 3–4 year period in
two cohort studies (Mayeux et al., 2003; Pomara et al., 2005). Conversely, low
baseline levels of plasma Aβ1–40 at age 77 years were associated with increased
AD risk in men (Sundelof et al., 2008). However, a recall for Aβ1–42 to be a
specific biochemical marker for AD has been more recently demonstrated (Uslu
et al., 2012).
. .. .. .. . .. : Plasma amyloid-β (Aβ) levels in sporadic Alzheimer’s disease
(AD) and control samples from a selection of cross sectional studies. Studies
were selected to show the range in plasma Aβ levels observed and the overlap
between AD and control cases. Data are presented as mean ± S.E. The sample
size for each group is given in parentheses. (a) Plasma Aβ40 levels, (b) Plasma
Aβ42 levels; SAD= sporadic AD (Reproduced from Bates et al., 2009).
.
Chapter One: Review of Literature
21
===================================================================
. .... ......
.. .. .. .. .
Cholesterol metabolism is an important element in AD risk and
pathogenesis, as evidenced by genetic, cell-culture, mouse model, and
epidemiologic data. High serum total cholesterol at midlife was linked to an
increased risk of late-life AD (Esiri et al., 1999; Fratiglioni et al., 2004). The
late-life high cholesterol in relation to dementia and AD is less clear, with
studies indicating either no association or an inverse association of
hypercholesterolemia with subsequent development of AD (Qiu et al., 2001;
Ngandu et al., 2007). A bidirectional influential relationship between serum
total cholesterol and dementia has been suggested; high total cholesterol at
middle age is a risk factor for the development of AD and dementia 20 years
later, but decreasing serum cholesterol after midlife may reflect ongoing disease
processes and may represent a marker for late-life AD and dementia (Fratiglioni
and Wang, 2000).
A pattern of decrease in blood pressure and BMI from midlife to older
adults has also been described, but decline in total cholesterol shows somewhat
different patterns. The dementia-associated additional decline in blood pressure
and BMI has been shown to become detectable about 3 to 6 years before the
clinical expression of the disease, while the decline in total cholesterol seems to
start much earlier, and with less evident acceleration prior to dementia onset
(Wang et al., 2009). These changes may explain, at least partly, the inconsistent
results from the cross-sectional and short-term follow-up studies, as well as
studies having the measurement of serum cholesterol later in life. In addition,
little information is currently available regarding the roles of cholesterol
subtypes (low-density lipoprotein, high-density lipoprotein, and triglycerides) in
AD (Povova et al., 2012). It has also been more recently reviewed that
cholesterol may impact Aβ production in the brain, and it has been
Chapter One: Review of Literature
22
===================================================================
demonstrated that dietary cholesterol increases amyloid production in rabbits,
and accordingly, the influence of cholesterol on APP processing and/or Aβ
generation has been subjected to an intensive investigation (Maulik et al.,
2013).
. .... ... ..........
... ... .. .. ....
Accumulating evidence suggests that brain tissues in AD patients are
exposed to oxidative stress during the development of the disease. Oxidative
stress or damage such as protein oxidation, lipid oxidation, DNA oxidation, and
glycoxidation is closely associated with the development of AD (Nunomura et
al., 2006). Oxidative stress is generally characterized by an imbalance in
production of reactive oxygen species (ROS) and antioxidative defense system,
which are responsible for the removal of ROS. Both systems are considered to
have major roles in the process of age-related neurodegeneration and cognitive
decline. Reactive oxygen species (ROS) and reactive nitrogen species (RNS),
including superoxide anion radical (O2−−), hydrogen peroxide (H2O2), hydroxyl
radical (•OH), singlet oxygen (1O2), alkoxyl radicals (•RO), peroxyl radicals
(ROO•), and peroxynitrites (ONOO−), contribute to pathogenesis of numerous
human degenerative diseases (Jung et al., 2009). Certain antioxidants including
glutathione, α-tocopherol (vitamin E), carotenoids, ascorbic acid, antioxidant
enzymes such as catalase and glutathione peroxidases are able to detoxify H 2O2
by converting it to O2 and H2O under physiological conditions. However, when
ROS levels exceed the removal capacity of antioxidant system under
pathological conditions or by aging or metabolic demand, oxidative stress
occurs and causes biological dysfunction. For example, high levels of protein
oxidation, lipid oxidation, advanced DNA oxidation and glycoxidation end
products, carbohydrates, formation of toxic substances such as peroxides,
Chapter One: Review of Literature
23
===================================================================
alcohols, aldehydes, free carbonyls, ketones, cholestenone, and oxidative
modifications in nuclear and mitochondrial DNA are the main manifestations of
oxidative stress or damage occurred during the course of AD (Lovell and
Markesber, 2007). Elevated levels of those oxidated formations mentioned
above were described not only in brain, but in cerebrospinal fluid (CSF), blood,
and urine of AD patients (Pratico et al., 2000).
Age-related memory impairments have also been correlated with a
decrease in brain and plasma antioxidants defense mechanism. An important
aspect of the antioxidant defense system is glutathione (GSH) which is
responsible for the endogenous redox potential in cells (Donahue et al., 2006).
Its most important function is to donate electrons to ROS so as to scavenge
them. Intracellular glutathione concentration decreases with age mammalian
brain regions including hippocampus, which may lead to a situation that the rate
of ROS production exceeds that of removal thus induces oxidative stress.
Therefore, the imbalance among the radical detoxifying enzymes is suggested
to be a cause for oxidative stress in AD (Zhu et al., 2006). Recently, Feng and
Wang (2012) indicated that oxidative stress not only strongly participates in an
early stage of AD prior to cytopathology, but plays an important role in
inducing and activating multiple cell signaling pathways that contribute to the
lesion formations of toxic substances and then promotes the development of
AD.
. .... .. ........
. .. .. .... .
C-reactive protein (CRP) is an acute-phase reactant that is synthesized by
the liver in response to acute injury, infection, or other inflammatory stimuli.
Prospective studies suggest that CRP levels in the highest tertile put one at
increased risk of developing cardiovascular disease. This risk holds for men,
Chapter One: Review of Literature
24
===================================================================
women, and the elderly population and does not appear to be moderated by race
or ethnicity (Ford and Giles, 2000; Tracy, 2002). As a result of this accumulated
evidence, the Centers for Disease Control and Prevention and the American
Heart Association presented interpretive guidelines for high-sensitivity CRP
(hs-CRP) with a cutoff score of <1.0 mg/L reflecting a low risk, 1.0 to 3.0 mg/L
reflecting an average risk, and >3.0 mg/L corresponding to a high risk in the
adult population. The highest risk tertile has approximately a 2-fold increased
risk of developing cardiovascular disease when compared to the lowest risk
tertile. Very highly elevated levels (>10 mg/L) may be due to noncardiovascular
causes of inflammation (Pearson et al., 2003). Inflammation has been shown to
play a role in cognitive decline, AD, and vascular dementia (Yaffe et al.,
2003;Engelhart et al., 2004; Dik et al., 2005).
There have been numerous studies linking CRP levels specifically to AD.
Schmidt et al. (2002) analyzed data from the Honolulu-aging study and
Honolulu-heart study and found that increased serum levels of CRP at midlife
were associated with increased risk of the development of AD, as well as
vascular dementia 25 years later. However, CRP levels did not predict AD
development in the Conselice Study of Brain Aging over a 4-year period
(Ravaglia et al., 2007). Similarly, over an average of a 5.7-year follow-up
period, CRP levels did not predict the development of AD among participants
(Van Oijen et al., 2005).
Cross-sectionally, very little data exist regarding serum CRP levels in
patients with established AD. A small study found that CRP levels were
elevated in AD and vascular dementia (Gupta et al., 2004). Locascio et al.
(2008) recently found that lower levels of CRP were associated with more rapid
cognitive and functional decline over time in patients diagnosed with AD. In a
more recent investigation, it has been demonstrated that midlife elevations in
Chapter One: Review of Literature
25
===================================================================
CRP are associated with increased risk of AD development though elevated
CRP levels were not useful for prediction in the immediate prodrome years
before AD becomes clinically manifest. However, for a subgroup of patients
with AD, elevated CRP continued to predict increased dementia severity, which
was suggestive of a possible pro-inflammatory endophenotype in AD (O’yry ant
et al., 2010).
. .... ......
.. .. ... ..... .. .
Alpha 1-Antitrypsin (α1-antitrypsin) is the major component of the alpha
band when serum is electrophoresed. Although the name implies that it acts
against trypsin, it is a general plasma inhibitor of proteases released from
leukocytes, especially elastase (Silverman et al., 2001). Elastase is an
endogenous enzyme that can degrade elastin and collagen. In chronic
pulmonary inflammation, lung tissue is damaged because of its activity. Thus,
α1-antitrypsin acts to counteract the effects of neutrophil invasion during an
inflammatory response (Spencer et al., 2004). It also regulates expression of
pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α),
interleukin-1 (IL-1), and IL-6 (Bosco et al., 2005). Alpha1-antitrypsin can also
react with any serine protease, such as those generated by triggering of the
complement cascade or fibrinolysis. Once bound to α1-antitrypsin, the protease
is completely inactivated and is subsequently removed from the area of tissue
damage and catabolized (Janciauskiene et al., 2004).
In AD, there is ample evidence for central and systemic activation of the
acute-phase response in patients with sporadic AD, although the significance of
this reaction in the pathophysiology of AD needs to be understood; however,
α1-antitrypsin levels have been reported to be elevated in the blood, brain and
CSF of AD patients (Puchades et al., 2003; Yu et al., 2003). Within the affected
Chapter One: Review of Literature
26
===================================================================
brain tissue, α1-antitrypsin protein was detected in astrocytes, senile plaques
and NFTs. Accordingly, it has been suggested that α1-antitrypsin and related
serpins of systemic or local (glial) origin may impact the natural history of AD
by suppressing Aβ fibrillogenesis, altering clearance of Aβ deposits within
senile plaques and serving as broad-spectrum inhibitors of AD-associated
neuroinflammation (Sun et al., 2003). Furthermore, Maes et al. (2006)
presented evidence of a direct and novel linkage between the acute-phase
response (α1-antitrypsin) and the dysregulation of central and peripheral
heme/iron/redox homeostasis that has been documented in patients with
sporadic AD.
. .... .. . . ... . .. ......
.... .. ... . .. .....
. ..
The human immunoglobulins are a family of proteins that confer humoral
immunity and perform vital roles in promoting cellular immunity. Five distinct
classes or isotypes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM) have
been identified in human serum on the basis of their structural, biological, and
antigenic differences. Immunoglobulin G and IgA have been further subdivided
into subclasses; IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively
on the basis of unique antigenic determinants (Normansell, 1987). Multiple
allotypic determinants in the constant region domains of human IgG and IgA
molecules as well as kappa (κ) light chains indicate inherited genetic markers.
In addition, there are several immunoglobulin-associated polypeptides such as
secretory component (SC) and J chain that have no structural homology with
the immunoglobulins, but serve important functions in immunoglobulin
polymerization and transport across membranes into a variety of secretions
(e.g., saliva, sweat, nasal secretions, breast milk, and colostrum). This diversity
of the immunoglobulin components of the humoral immune system provides a
Chapter One: Review of Literature
27
===================================================================
complex network of protective and surveillance functions (Torres and
Casadevall, 2008). From a clinical perspective, quantitative levels of these
analytes in serum can aid in the diagnosis and management of
immunodeficiency, abnormal protein metabolism, and malignant states. As
such, they provide a differential diagnosis as to possible causes of recurrent
infections and can indicate a strategy or subsequent therapeutic intervention
(Stevens, 2010), especially when we consider that their level in humans is
influenced by multiple factors, and age is possibly the most important personal
attribute that determines serum immunoglobulin levels. Therefore, serum
immunoglobulin level is worth to be investigated in AD, because age is one
important risk factor for the disease (Matthews et al., 2009). However, few
studies have examined serum immunoglobulin levels in AD patients and most
of them were carried out in the 1980s. In an earlier study, it was demonstrated
that the majority of pre-senile AD patients showed a reduced serum level of one
or more of the immunoglobulins IgG, IgA or IgM (Pentland et al., 1982). A
further study examined the plasma level of IgG in 20 patients with Alzheimer's
dementia or senile dementia of Alzheimer type (AD/SDAT), 23 with multiinfarct dementia (MID) and 16 controls, and found that MID patients had
significantly elevated plasma IgG levels compared to controls and AD/SDAT
patients (Alafuzoff et al., 1983). However, three later studies reported no
significant differences between AD patients and controls in the serum or plasma
level of one or more of the three immunoglobulin classes (Elovaara, 1984;
Elovaara et al., 1987; Kay et al., 1987).
. .... ... . ... . .. .... . .. ... ..... .......
.
The complement system represents a complex and tightly regulated attack
system designed to destroy invaders and to assist in the phagocytosis of waste
Chapter One: Review of Literature
28
===================================================================
materials. The components of this system carry out four major functions:
recognition, opsonization, inflammatory stimulation, and direct killing through
the membrane attack complex (McGeer and McGeer, 2002). Complement
proteins interact with cell surface receptors to promote a local inflammatory
response that contributes to the protection and healing of the host. Complement
activation causes inflammation and cell damage, yet it is essential for
eliminating cell debris and potentially toxic protein aggregates (Shen and Meri,
2003). The complement system consists of more than 30 fluid-phase and cellmembrane-associated proteins that can be activated by different routes
(classical, lectin and alternative pathways). The classical pathway (involving
C1q, C1r, C1s, C4, C2, and C3 components) is activated primarily by the
interaction of C1q with immune complexes, but activation can also be achieved
after interaction of C1q with non-immune molecules such as DNA, RNA, Creactive protein, serum Aβ, bacterial lipopolysaccharides, and some fungal and
virus membranes (Bohlson et al., 2007).
The possible role of the complement system in AD has been frequently
discussed, and accumulating evidences suggested that such system is fully
activated in AD. Complement proteins of both the classical and alternative
pathways (such as C1q, C4, C3, and Factor B) have been colocalized with
fibrillar amyloid plaques and cerebral vascular amyloid in the cerebral cortex
and hippocampus of AD patients (Stoltzner et al. 2000). The C5b-9 membrane
attack complex has been found associated with myelin and membranes in AD
brain, demonstrating that in this disorder the entire complement cascade is
activated, and in vitro, Aβ fibrils (fAβ) have been shown to activate both the
classical complement pathway by directly binding to C1q and the alternative
pathway via interactions with C3 (Zhou et al., 2008). Thus, it was hypothesized
that in vivo fAβ activates the complement cascade and contributes to local
Chapter One: Review of Literature
29
===================================================================
inflammation, particularly by recruiting glial cells into the area of the plaque,
resulting in neurotoxicity and dementia (McGeer and McGeer, 2010); therefore,
determining the serum level of C3 and C4 in AD patients may have potential to
understand the pathology of disease.
.. . ..... ... .. ..
Cytokines are small soluble proteins (MW: 8-40 kDa) that regulate the
immune system, orchestrating both innate immunity and the adaptive responses.
They are secreted by a variety of immune cells (e.g., T-lymphocytes,
macrophages, natural killer cells) and non-immune cells (e.g., Schwann cells,
fibroblasts). The biological effects induced by cytokines include stimulation or
inhibition of cell proliferation, cytotoxicity/apoptosis, antiviral activity, cell
growth and differentiation, inflammatory responses, and up-regulation of
expression of surface membrane proteins (Commins et al., 2010). These effects
are achieved through both autocrine stimulation (i.e., affecting the same cell
that secreted it) and paracrine (i.e., affecting a target cell in close proximity)
activities, and can also exert systemic or endocrine activities. The main function
of cytokines is the regulation of T-cell differentiation from undifferentiated
cells to T-helper 1 and 2, regulatory T cells, and T-helper 17 cells. These
regulatory proteins include interleukins (ILs), interferons (IFNs), colony
stimulating factors (CSFs), tumor necrosis factors (TNFs), and certain growth
factors (Babon and Nicola, 2012). Cytokines are induced in response to specific
stimuli; for instance, bacterial lipopolysaccharides, flagellin, or other bacterial
products, through the ligation of cell adhesion molecules or through the
recognition of foreign antigens by host lymphocytes (Broughton et al., 2012).
Many of these cytokines have been shown to be produced by neurons or
glia and there are a number of reports indicating changes in their levels in AD
Chapter One: Review of Literature
30
===================================================================
brain, blood and cerebrospinal fluid (Weisman et al., 2006). Levels of IL-1α,
IL-1β, LL-6, TNF-α and IFN-αhave been reported to be increased in AD
patients, and a number of interactions between cytokines and components of the
AD senile plaques have also been reported suggesting that a vicious circle
might be generated (McGeer and McGeer, 2010). Thus, the Aβ protein of the
plaques has been suggested to potentiate the secretion of several interleukins by
activated astrocytoma cells; moreover, synergistic effects may also occur
between cytokines and Aβ. For example, IFN-γ has been shown to synergize
with Aβ to cause the release of TNF-α and reactive nitrogen species that are
toxic to neurons, and IL-1 is reported to increase the toxicity of Aβ in CC12 cell
line (Griffin and Barger, 2010).
In the present study, three cytokines were investigated: IL-1α, IL-10 and
IL-17A.
. ... . ... . .... .... .. .αα.
Interleukin-1 and its related family members are primarily proinflammatory cytokines by their ability to stimulate the expression of genes
associated with inflammation and autoimmune diseases. The most salient and
relevant properties of IL-1 in inflammation are the initiation of cyclooxygenase
type 2 (COX-2), type 2 phospholipase A and inducible nitric oxide synthase
(iNOS). This accounts for the large amount of prostaglandin-E2 (PGE2),
platelet activating factor and nitric oxide (NO) produced by cells exposed to IL1 or in animals or humans injected with IL-1 (Dinarello, 1997). Another
important pro-inflammatory property of IL-1 is its ability to increase the
expression of adhesion molecules, such as intercellular adhesion molecule-1
(ICAM-1), on mesenchymal cells and vascular-cell adhesion molecule- 1
(VCAM- 1) on endothelial cells. This latter property promotes the infiltration of
Chapter One: Review of Literature
31
===================================================================
inflammatory and immunocompetent cells into the extravascular space
(Dinarello, 1998).
The IL-1 family of cytokines includes two agonist proteins, IL-1α and IL1β, which trigger cell activation upon binding with specific membrane
receptors. Also included is IL-1 receptor antagonist (IL-1ra), which is a
glycosylated secretory protein of 23 kDa that counteracts the action of IL-1
(Griffin and Mrak, 2002). Interleukin-1α and IL-1β (MW of each: 17 kDa) are
produced by monocytes and macrophages, and can be induced by the presence
of microbial pathogens, bacterial lipopolysaccharides, or other cytokines. IL-1α
and IL-1β exhibit the same activities in many test systems and share
approximately 30% sequence homology. However, IL-1α remains intracellular
within monocytes and macrophages and is rarely found outside these cells, but
can be released after cell death and can promote the attraction of inflammatory
cells to areas where cells and tissues are being killed or damaged (Basu et al.,
2004).
The first assessment of IL-1 in sera of AD patients was carried in 1991 by
Cacabelos and co-workers (Cacabelos et al., 1999). The authors demonstrated
that serum IL-1α levels did not differ significantly between healthy elderly
subjects, early-onset AD, late-onset AD or MID patients, but, a negative
correlation between mental performance, IL-1α and IL-1β was observed in lateonset AD patients. Later studies revealed the importance of IL-1α in pathogenesis of AD, especially in connection with DS (Konstantinos et al., 2008).
Microglial cells were found to express excessive amounts of the systemic
immune response-generating cytokine IL-1 in the AD brain. Considering the
relationships between DS and AD, the possibility that changes that are nearly
universal in the DS brain would render it a suitable system for studying drivers
of AD pathogenesis was explored. Compared with the brains of non-DS
Chapter One: Review of Literature
32
===================================================================
individuals of similar ages, IL-1 was found to be highly overexpressed in
activated glia, even in the brains of fetuses, neonates, and children with DS
(Griffin and Mrak, 2002). This age distribution was important; because neither
Aβ plaques nor NFTs were noted in these brains, nor have these AD
neuropathological anomalies been reported in brains from DS individuals of
these ages. Accordingly, it was found that an increase in IL-1 expression was
observed in DS prior to the detection of either Aβ plaques or NFTs. In addition,
the capacity of IL-1to elevate neuronal βAPP, the induction of tau
phosphorylation through IL-1-induced activation and the relation of
overexpression of IL-1 to both Aβ plaque and NTFs development were
observed. These events can also promote for AD-related symptoms; including
tau pathology and inhibition of neurogenesis by IL-1 (Griffin and Barger,
2010).
. ... . ... . .... .... .. ... .
Interleukin-10 was discovered in 1989 and was originally characterized by
its ability to inhibit the production of pro-inflammatory cytokines by
macrophages and TH1 cells. However, IL-10 is also recognized to directly affect
the growth and development of a variety of cells. For example, IL-10 can
directly promote the death of inflammatory cells, including activated
macrophages. In contrast, IL-10 also promotes the survival of many types of
cells via different mechanisms (Moore et al., 2001). Various cell populations
including certain T cell subsets, monocytes, and macrophages have the capacity
for IL-10 production. Thus macrophages, the major source of IL-10, are
stimulated to produce IL-10 by several endogenous and exogenous factors such
as endotoxins and TNF-α (Platzer et al., 2000). On the other hand, systemic
release of TNF-α also induces LL-10 via a negative feedback using NF-jB-
Chapter One: Review of Literature
33
===================================================================
dependent pathway (Ma et al., 2001). Interleukin-10 is a pleiotropic cytokine
that inhibits cell mediated immunity while enhancing humoral immunity. It also
inhibits the synthesis of a number of cytokines such as IFN-,, LL-2, and TNF-α.
It also appears that IL-10 is a neutralizing component of inflammation and serves
to reduce both duration and magnitude of the process, and IL-10 gene-deficient
mice show overproduction of inflammatory cytokines and the development of
chronic inflammatory disease (Kaur et al., 2009).
Due to its anti-inflammatory properties, IL-10 has also been a subject of
investigation in AD patients. Interlukin-10 mRNA has been detected in the
frontal and parietal lobe of the normal brain, and has been suggested to play an
important role in neuronal homeostasis and cell survival through interacting with
specific cell surface receptors (IL-10Rs), present on all the major glial cell
populations in the brain (Strle et al., 2001), and it limits inflammation by
reducing the synthesis of pro-inflammatory cytokines such as IL-1 and TNF-α,
by suppressing cytokine receptor expression and by inhibiting receptor activation
in the brain (Ledeboer et al., 2002). It has also been demonstrated that Aβ was
not able to stimulate IL-10 production by glial cells in vitro, but pre-exposure of
glial cells to IL-10 inhibits Aβ- or LPS-induced production of pro-inflammatory
cytokines, and accordingly, glia cells and their cytokines may contribute in the
progression of neurodegeneration in AD patients (Mrak and Griffin, 2005).
These findings have suggested to hypothesis that IL-10 and its genetic
polymorphism can affect the risk of developing late onset AD (Di Bona et al.,
2012).
. ... . ... . .... .... .. .. . . .
Interleukin-17A was discovered in 1993 originally as a rodent T cell
cDNA transcript, cytotoxic T lymphocyte-associated antigen 8 (CTLA8), and
Chapter One: Review of Literature
34
===================================================================
subsequently human IL-17A was identified (Yao et al., 1995). To date, five
additional members of the IL-17 family have been identified and termed IL17B, IL-17C, IL-17D, IL-17E and IL-17F. IL-17F is most closely related to IL17A, and can form a heterodimer with IL-17A, while IL-17E, also named IL25, is instead classified as a TH2 cytokine. There are five receptors for the IL-17
family of cytokines (IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE), of
which IL-17RA and IL-17RC mediate the biologic activity of IL-17A. While
IL-17A is produced mainly by T cells, its receptor is expressed ubiquitously on
various cell types, including myeloid cells, epithelial cells, and fibroblasts.
Therefore, IL-17A exerts various biological functions in vivo, which might be
involved in the pathogenesis of a wide range of inflammatory disorders, as well
as, infectious conditions (Iwakura et al., 2011).
Interleukin-17A is produced mainly by TH17 cells, but other cells (CD8+T
cells, δδ T cells, NK cells, activated monocytes and neutrophils) are also able to
produce it (Santarlasci et al., 2009). It triggers pro-inflammatory responses, and
upon receptor binding, it induces expression of multiple genes involved in tissuemediated innate immunity including pro-inflammatory chemokines (CXCL1,
CXCL8, CXCL10), cytokines (TNF-α, LL-1, IL-6, GM-CSF, G-CSF),
antimicrobial peptides (mucins, β-defensins and S100A7-9), and proteins
involved in tissue remodeling and acute phase responses (serum amyloid A,
matrix metalloproteinase [MMP]-1 and receptor activator of NF-BB ligand)
(Yamada, 2010). Over-expression of IL-17A in vivo has been shown to increase
neutrophil infiltration through modulation of pro-inflammatory cytokines and
chemokines resulting in inflammation (Iwakura et al., 2011). These biological
effects prompted the present study to investigate IL-17A in AD, but for the best
knowledge of the investigator, this cytokine has not been investigated in AD.
Chapter Two: Subjects, Materials and Methods
۳٥
===================================================================
Chapter Two
Subjects, Materials and Methods
2.1 Patients and Controls
Three groups of subjects were enrolled in the present study during the
period November 2011 - May 2012. The first included 30 cases of Alzheimer's
disease (AD), with an age range of 38-100 years. These cases were ascertained
through Psychiatric Private Clinics distributed in Baghdad and surrounding
governorates, in which the diagnosis was made. The diagnosis was based on the
National Institute of Neurological and Communicative Disorders and Stroke
and the Alzheimer's Disease and Related Disorders Association (now known as
the Alzheimer's Association) work-group criteria (NINCDS-ADRDA). This
diagnostic tool speciifes eight cognitive domains that may be impaired in AD:�
memory, language, perceptual skills, attention, constructive abilities,
orientation, problem solving and functional abilities (Dubois et al., 2007). In
addition, all patients were evaluated using Magnetic Resonance Imaging (MRI)
to reach the most probable clinical diagnosis of AD. The second group included
28 patients (age range: 61-92 years), who had vascular dementia (VD), and they
were ascertained from the same clinics. Both groups of patients were subjected
to a personal interview using a designed questionnaire (Appendix I). A third
group included 10 Down’s syndrome (DS) cases with an age range of �-15
years.
A fourth group of age (age range: 44-90 years), gender and ethnicity (Arab
Muslims) matched controls were also enrolled. They were 20 individuals who
had no history of any cognitive difficulties (apparently healthy.).
Chapter Two: Subjects, Materials and Methods
۳٦
===================================================================
2.2 Materials
2.2.1 Equipment, Plastic and Glassware
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Cecil E1021 spectrophotometer (England).
Centrifuge: Beckman (Germany).
Conical flasks: Aldrich (Germany).
Deep Freezer (-20°C): Ishtar (Iraq).
Disposable syringes (5ml): Medico (United Arab Emirates).
EDTA tubes: Plasti LAB (Lebanon).
ELISA System: Human Reader (Germany).
Eppendorf tubes: Grenier (Germany).
Graduated cylinder (25ml, 50ml, 100ml and 1000ml): MBL (UK).
Graduated plain tube (10ml): AFMA (Jordon).
Pipette tips: Gilson (France).
Pipette: Gilson (France).
Plastic disposable pipette (1.5mm pore): Gilson (France).
Plate washer: Human Reader (Germany).
Precision pipettes (20µl, 50µl, 100µl and 1000 µl): Gilson (France).
Refrigerator: Arcelik (Turkey).
Shaker: Rotal (England).
Shaking water bath: GFI (Germany).
Vortex: Retch (Germany).
Water bath: Beckman (Germany).
2.2.2 Laboratory Kits
• Cholesterol determination kit: Human (Germany).
• Enzyme Immunoassay kit for the detection of human beta amyloid
protein: USBio (USA).
• HDL determination kit: Human (Germany).
• High sensitive C-reactive protein kit: Demeditec (Germany).
• Human IL-10 ELISA Development kit: PEPROTECH (USA).
• Human IL-17 ELISA Development kit: PEPROTECH (USA).
• Human IL-1α ELISA Development kit: PEPROTICH (USA).
• Single Radial Immunodiffusion plates for determination of
immunoglobulins (IgA, IgG and IgM) and complement components
C3 and C4: LTA (Italy).
• Total antioxidant capacity kit: USBio (USA).
Chapter Two: Subjects, Materials and Methods
۳۷
===================================================================
• Triglyceride determination kit: Human (Germany).
2.3 Collection of Blood Samples
From each participating subject, 3-5 ml of blood was obtained by
venipuncture. The collected blood was transferred to a plain tube and left to clot
at room temperature (20-25°C) for 15 minutes. The clotted blood was
centrifuged (2000 rpm) for 15 minutes; and by then, serum was collected and
distributed into aliquots of 0.25 ml in Eppendorf tubes, which were frozen at 20°C until laboratory assessments (Dacie and lewis,2005).
2.4 Laboratory Methods
2.4.1 Beta Amyloid1-40 (Aβ1-40) Assessment
A. Principles of the Test
The human Aβ1-40 kit is a solid phase sandwich ELISA, in which a monoclonal
antibody specific for the NH2-terminus of human Aβ1-40 was coated onto the
wells of microtiter strips. During the first incubation, standards of know human
Aβ1-40 concentrations and samples (serum) are pipetted into the wells and coincubated with a rabbit antibody specific for the COOH-terminus of the human
Aβ1-40. Bound rabbit antibody is detected by the use of a horseradish
peroxidase-labeled (HRP) anti-rabbit antibody, which is added after a washing
step. After a second incubation and washing to remove the unbound enzyme, a
substrate solution is added, which acts upon by the bound enzyme to produce a
color. The intensity of this colored product is directly proportional to the level
of human Aβ1-40 present in standards and serum samples (Kruman,2002).
B. Kit Contents
• Human Aß1-40 standard.
• Standard diluent buffer.
Chapter Two: Subjects, Materials and Methods
۳۸
===================================================================
• Pre-coated 96 well plate with mAb to NH2-terminus of Aß1-40.
• Rabbit anti-human Aß1-40 detection antibody.
• Anti-rabbit IgG HRP (Horseradish Peroxidase).
• HRP diluent.
• Wash buffer concentrate.
• Stabilized chromogen: Tetramethylbenzidine (TMB).
• Stop solution.
C. Assay Procedure
Before carrying out the assay procedure of Aß 1-40 determination, the kit
was left at room temperature (18-25ºC) for 30 minutes to equilibrate, as
suggested by the manufacturer. After that assay was carried out following the
instructions in the kit's leaflet (USBio; USA), which are summarized in the
following steps:
i. Standard diluent buffer (50 μl) was first added to each well, followed by
50 μl of standards (0, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 pg/ml) or
samples to the respective wells, followed by 50 μl of rabbit anti-human
Aß1-40 detection antibody. The plate was covered with its cover and
incubated at room temperature (20 to 25°C) for 3 hours on a microplate
shaker set at 200 rpm.
ii. The well contents were aspirated and decanted on filter paper, and each
well was washed four times with washing buffer. After that, 100 μl of antirabbit IgG HRP was added to all wells, and the plate was covered with its
cover and incubated for 30 minutes at room temperature on a microplate
shaker set at 200 rpm.
iii. The washing step was repeated, followed by adding 100 μl Stabilized
chromogen, and the plate was incubated for 30 minutes in a dark place.
Then, stop solution (1M Phosphoric acid; 100 μl) was added to all wells,
and absorbance was read at wave length of 450 nm using ELISA reader.
Chapter Two: Subjects, Materials and Methods
۳۹
===================================================================
iv. The sample results were calculated by interpolation from a standard curve
that was performed in the same assay as that for the samples by using a
standard curve fitting equation (Figure 2-1).
4.5
y = 0.0082x + 0.0031
R2 = 0.9926
4.0
Absorbance (450 nm)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0
100
200
300
400
500
600
Serum Level of Beta Amyloid (pg/ml)
Figure 2-1: Standard curve of beta amyloid.
2.4.2 Cholesterol Determination (Schettler,et al.,1975)
The serum level of cholesterol was determined after enzymatic hydrolysis
and oxidation, and the indicator quinoneimine is formed from hydrogen
peroxide and 4-aminophenazone in the presence of phenol and peroxidase.
Based on instructions of the kit manufacturer (Human; Germany), the following
pipetting scheme was applied(Schettler,et al.,1975).
Tubes
Reagent
Sample
Standard
1000 µl
10 µl
10 µl
-
1000 µl
1000 µl
The contents of each tube were mixed and incubated at room temperature
(18-25ºC) for 10 minutes, and by then, the absorbance of sample and standard
Chapter Two: Subjects, Materials and Methods
٤۰
===================================================================
was read at a wave length of 500 nm against the reagent blank. The serum level
of cholesterol was calculated by the following equation:
 Absorbance of Sample 
Serum Level of Cholesterol (mg/dL) = 200 x 

 Absorbance of Standard 
2.4.3 Triglycerides Determination
The serum level of cholesterol was determined after enzymatic hydrolysis
with lipases, and the indicator quinoneimine is formed from hydrogen peroxide,
4-aminoantipyrine and 4-chlorophenol under the catalytic influence of
peroxidase (Kit leaflet: Human; Germany). The pipetting scheme and
calculations were as that of cholesterol (Section 3.4.2) (Schettler,et al.,1975).
2.4.4 High Density Lipoproteins (HDL) Cholesterol
The chylomicrons, VLDL (very low density lipoproteins) and LDL (low density
lipoprotein) in the serum were precipitated by addition of 500 µl of precipitant
(phosphotungstic acid and magnesium chloride) to 200 µl of serum in a plain
tube. The tube contents were mixed and incubated for 10 minutes at room
temperature (18-25ºC). The tube was centrifuged (6000 rpm for 10 minutes),
and the supernatant (contains the HDL fraction) was collected and assayed for
HDL cholesterol with the cholesterol kit(Gordon,et al.,1977). Based on
instructions of the kit manufacturer (Human; Germany), the following pipetting
scheme was applied:
Tubes
Reagent
Standard
Sample
100 µl
-
-
Standard
-
100 µl
-
HDL supernatant
-
-
100 µl
1000 µl
1000 µl
1000 µl
Distilled water
Reagent
Chapter Two: Subjects, Materials and Methods
٤۱
===================================================================
The contents of each tube were mixed and incubated at room temperature
(18-25ºC) for 10 minutes, and by then, the absorbance of sample and standard
was read at a wave length of 500 nm against the reagent blank. The serum level
of HDL cholesterol was calculated by the following equation:
 Absorbance of Sample 
Serum Level of HDL Cholesterol (mg/dL) = 150 x 

 Absorbance of Standard 
2.4.5 Low Density Lipoproteins (LDL) Cholesterol
The serum level of LDL cholesterol was calculated from the levels of
cholesterol, HDL cholesterol and triglycerides according to an equation
presented by Friedewald et al. (1972), and as the following:
 Triglycerides 
LDL Cholesterol (mg/dL) = Cholesterol - HDL Cholesterol x 

5


2.4.6 Very Low Density Lipoproteins (VLDL) Cholesterol
The serum level of VLDL cholesterol was calculated from the levels of
triglycerides according to an equation presented by Friedewald et al. (1972),
and as the following:
 Triglycerides 
VLDL Cholesterol (mg/dL) = 

5


2.4.7 Total Antioxidant Capacity
A. Background of Test
Antioxidants play an important role in preventing the formation of and
scavenging of free radicals and other potentially toxic oxidizing species. There
are three categories of antioxidant species: enzyme systems (GSH reductase,
catalase, peroxidase, etc.), small molecules (ascorbate, uric acid, GSH, vitamin
E, etc.) and proteins (albumin, transferrin, etc.). Different antioxidants vary in
their reducing power. Trolox is used to standardize antioxidants, with all other
antioxidants being measured in Trolox equivalents. Measurement of the
Chapter Two: Subjects, Materials and Methods
٤۲
===================================================================
combined non-enzymatic antioxidant capacity of biological fluids and other
samples provides an indication of the overall capability to counteract reactive
oxygen species (ROS), resist oxidative damage and combat oxidative stress
related diseases. In some cases, the antioxidant contribution of proteins is
desired whereas in other cases only the contribution of the small molecule
antioxidants is needed. The Total Antioxidant Capacity Assay Kit can measure
either the combination of both small molecule antioxidants and proteins or
small molecules alone in the presence of proprietary Protein Mask. Cu ++ ion is
converted to Cu+ by both small molecule and protein. The Protein Mask
prevents Cu++ reduction by protein, enabling the analysis of only the small
molecule antioxidants. The reduced Cu+ ion is chelated with a colorimetric
probe giving a broad absorbance peak around 570 nm, proportional to the total
antioxidant capacity (Kit leaflet: USBio; USA) (Neurochem,2002).
B. Kit Content
• Cu++ Reagent.
• Assay Diluent.
• Protein Mask.
• Trolox Standard.
C. Assay Procedure
First, the trolox standards were prepared by adding 0, 4, 8, 12, 16, 20 μl
of the Trolox standard to individual wells of 96-well ELISA plate, and then the
volume of each well was adjusted to 100 µl with doubled distilled water to give
0, 4, 8, 12, 16, 20 nmol of Trolox standard. Second, 100 µl of each tested
sample (serum) were added to the other wells. Finally, 100 µl of Cu++ Reagent
working solution were added to all wells, and the plate was covered and
Chapter Two: Subjects, Materials and Methods
٤۳
===================================================================
incubated at room temperature (18-25ºC) for 1.5 hours. After that, the
absorbance of each well was read at 570 nm using ELISA reader.
The sample results were calculated by interpolation from a standard curve
that was performed in the same assay as that for the samples by using a standard
curve fitting equation for total antioxidant capacity (Figure 2-2).
0.8
y = 0.0317x + 0.0943
R2 = 0.9424
Absorbance (570 nm)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
5
10
15
20
25
Total Antioxidant Capacity (nmol)
Figure 2-2: Standard curve of total antioxidant capacity.
2.4.8 High Sensitive C-reactive Protein (hsCRP)
A. Principles of the Test
The human hsCRP kit is based on ELISA principles, in which a
monoclonal antibody specific for human hsCRP was coated onto wells of
microtiter strips. During incubation, CRP in standards or samples (serum)
binds specifically to the wells. After removal of unbound serum proteins by
washing, the antigen-antibody complex in each well is detected with a specific
peroxidase-conjugated antibody. After removal of the unbound conjugate, the
wells are incubated with a chromogen solution containing TMB and hydrogen
peroxidase, and by then a blue color develops in proportion to the amount of
Chapter Two: Subjects, Materials and Methods
٤٤
===================================================================
immune-complex bound to the wells. The enzymatic reaction is stopped by
the addition of stop solution and the absorbance is read at a wave length of
450 nm. (Mitra and Panja,2005).
B. Kit Contents
• Micro-titer-strip wells (8 x 12) coated with monoclonal anti-human CRP
antibodies.
• Standard CRP.
• Conjugate.
• Dilution buffer.
• Washing solution.
• Chromogen solution (H2O2 + TMB).
• Stop solution.
C. Assay Procedure
Before carrying out the assay procedure of hsCRP determination, the kit
was left at room temperature (18-25ºC) for 30 minutes to equilibrate, as
suggested by the manufacturer. After that the assay was carried out following
the instructions in the kit's leaflet (Demeditec; Germany), which are
summarized in the following steps:
i. The sera of patients were first diluted (1:1000) with dilution buffer, and
then 100 µl of standards (0, 0.4, 1, 5 and10 μg/ml) and diluted sera were
pipetted in the respective wells, and the plate was covered and incubated
for 30 minutes at room temperature (18-25ºC).
ii. The wells were washed four times (washing solution), and then 100 µl of
conjugate solution was added to each well. The plate was covered and
incubated for 30 minutes at room temperature (18-25ºC).
iii. The washing step was repeated, followed by adding 100 μl of chromogen
solution, and the plate was incubated for 10 minutes in a dark place. Then,
Chapter Two: Subjects, Materials and Methods
٤٥
===================================================================
stop solution (50 μl) was added to all wells, and absorbance was read at
wave length of 450 nm using ELISA reader.
iv. The sample results were calculated by interpolation from a standard curve
that was performed in the same assay as that for the samples by using a
standard curve fitting equation (Figure 2-3).
2.5
y = 0.1973x + 0.031
R2 = 0.9997
Absorbance (450 nm)
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
C-reactive Protein (μg/ml)
Figure 2-3: Standard curve of high sensitive C-reactive protein.
2.4.9 Alpha1-antitrypsin, Immunoglobulins and Complement
A. Principle of Assay
The total serum level of α1-antitrypsin, immunoglobulins (IgA, IgG and
IgM) and complement components C3 and C4 was determined by means of
Single Radial Immunodiffusion Assay. It is a single radial immunodiffusion
test, which was developed by Mancini et al. (1965) for quantitive determination
of proteins in the serum. Test sample is added to a well in an agarose gel
containing a monospecific antiserum. The sample diffuses radially through the
gel and the substance being assayed forms a precipitation ring with the
Chapter Two: Subjects, Materials and Methods
٤٦
===================================================================
monospecific antiserum. Ring diameter is measured and the concentration is
determined from the reference standard curve.
B. Procedure
Before starting the assay, the plates were opened and left for 5 minutes at
room temperature (18-25ºC), and then 5 µl of serum was dispensed into a well
in the plate. The plate was incubated in flat position at room temperature for 72
hours (α1-antitrypsin, IgA, IgG, C3 and C4) or 96 hours (IgM). The ring
diameter was measured by an ocular and the concentration was obtained from
the reference curve.
2.4.10
Cytokines (IL-1α, IL-10 and IL-17A) (www.peprotech.com)
The sera of patients and controls were assessed for the level of three
cytokines, which were IL-1α, IL-10 and IL-17A by means of ELISA that were
based on similar principles.
A. Principles of Assay
The human IL-1α, IL-10 or IL-17A kit (PeproTech; USA) is a sandwich
enzyme-linked immunosorbent assay designed for the quantitative measurement
of natural or recombinant human IL-1α, IL-10 or IL-17A in serum, plasma and
other biological fluids, in which an anti-human IL-1α, IL-10 or IL-17A coating
antibody (Capture Antibody) is adsorbed onto wells of 96-well plate. Human
cytokine present in sample or standard binds to antibodies that were adsorbed to
the wells. A biotinylated anti-human cytokine antibody is added and binds to
human cytokine captured by the first antibody (Detection Antibody). Following
incubation, unbound biotinylated anti-human cytokine antibody is removed
during a wash step. Avidin horse-radish peroxidase (HRP) conjugate is then
added and binds to the biotinylated antihuman cytokine antibody. Following
Chapter Two: Subjects, Materials and Methods
٤۷
===================================================================
incubation, unbound avidin-HRP conjugate is removed during a wash step, and
a substrate solution reactive with HRP is added to the wells. A colored product
is formed in proportion to the amount of human cytokine present in the sample
or standard. The color development is monitored with ELISA plate reader and
absorbance is measured at wave length of 405 nm. A standard curve is prepared
from standard dilutions and human cytokine sample concentration is determined
from a curve fitting equation.
A. Kit Contents
• ELISA plate: Blank 96-well plate
• Capture antibody: Goat anti-human IL-1α, IL-10 or IL-17A antibody.
• Detection antibody: Biotinylated anti-human IL-1α, IL-10 or IL-17A
antibody.
• Standards: Recombinant human IL-1α, IL-10 or IL-17A.
• Avidin-HRP conjugate.
• ABTS liquid substrate solution.
• Washing buffer: 0.05% Tween-20 in phosphate buffer saline (PBS).
• Block buffer: 1% bovine serum albumin (BSA) in PBS.
• Diluent: 0.05% Tween-20 and 1% BSA in PBS.
B. Assay Procedure
Before carrying out the assay procedure of IL-1α, IL-10 or IL-17A
determination, the kit was left at room temperature (18-25ºC) for 30 minutes to
equilibrate, as suggested by the manufacturer. After that assay was carried out
following the instructions in the kit's leaflet (PeproTech; USA), which are
summarized in the following steps:
Chapter Two: Subjects, Materials and Methods
٤۸
===================================================================
i. The wells of plate were coated with capture antibody by dispensing 100 µl
of anti-human IL-1α, IL-10 or IL-17A antibody in each well, and the
plate was sealed and incubated overnight at room temperature (18-25ºC).
ii. The day after, the contents of wells were discarded and each well was
washed four times with washing buffer (300 µl/well/wash), and then the
plate was inverted to remove residual buffer and blotted on towel paper.
iii. In each well, 100 µl of block buffer was dispensed and the plate was
incubated at room temperature for 60 minutes, and then the washing step
was repeated (step ii).
iv. An aliquot (100 µl) of standards of IL-1α (3.9, 7.8, 15.6, 31.2, 62.5, 125,
500 and 1000 pg/ml), IL-10 (19.5, 39.1, 78.13, 156.25, 321.5, 625, 1250
and 2500 pg/ml) or IL-17A (7.8, 15.6, 31.2, 62.5, 125, 500, 1000 and
2000 pg/ml) or serum samples was dispensed into the respective wells.
The plate was incubated at room temperature for two hours, and then the
washing step was repeated (step ii).
v. An aliquot (100 µl) of detection antibody (biotinylated anti-human IL-1α,
IL-10 or IL-17A antibody) was dispensed in each well. The plate was
incubated at room temperature for two hours, and then the washing step
was repeated (step ii).
vi. An aliquot (100 µl) of avidin-HRP conjugate was dispensed in each well.
The plate was incubated at room temperature for 30 minutes, and then the
washing step was repeated (step ii). Finally, 100 µl of substrate solution
was added, and color development was monitored with ELISA plate
reader and absorbance was measured at a wave length of 405 nm. Three
readings were done (3, 6, and 9 minutes) and the mean absorbance was
considered for calculations.
Chapter Two: Subjects, Materials and Methods
٤۹
===================================================================
vii. The sample results were calculated by interpolation from a standard curve
that was performed in the same assay as that for the samples by using a
standard curve fitting equation (Figures 2-4, 2-5 and 2-6 for IL-1α, IL-10
and IL-17A, respectively).
0.8
y = 0.0006x + 0.0803
R2 = 0.9043
Absorbance (405 nm)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
200
400
600
800
IL-1α Level (pg/ml)
Figure 2-4: Standard curve of IL-1α.
1000
1200
Chapter Two: Subjects, Materials and Methods
٥۰
===================================================================
1.4
y = 0.0005x + 0.1863
2
R = 0.906
Absorbance (405 nm)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
500
1000
1500
2000
2500
3000
IL-10 Level (pg/ml)
Figure 2-5: Standard curve of IL-10.
1.2
y = 0.0005x + 0.0421
R2 = 0.9914
Absorbance (405 nm)
1.0
0.8
0.6
0.4
0.2
0.0
0
500
1000
1500
2000
IL-17A Level (pg/ml)
Figure 2-6: Standard curve of IL-17A.
2500
Chapter Two: Subjects, Materials and Methods
٥۱
===================================================================
2.5 Statistical analysis
Data were presented as either percentage frequencies or means ± standard
errors (S.E.). Significant differences between percentage frequencies were
assessed by Pearson’s Chi-square test, while such differences between means
were assessed by ANOVA (analysis of variance) followed by Duncan test, in
which probability (P) ≤ 0.05 was considered significant. In both cases, the
computer package SPSS version 16 was used to carry out such analysis. In
further analyses, significant differences between proportions were assessed by Z
test. Odds ratio was also assessed in some cases. The latter two assemments
were carried out using the computer package PEPI version 4.
Chapter Three: Results and Discussion
٥۲
================================================================
Chapter Three
Results and Discussion
3.1 Demographic Presentation of Study Groups
The results presented in this study were based on analyses of data from
a total of 88 cases: 30 Alzheimer's disease (AD), 28 vascular dementia (VD),
10 Down's syndrome (DS), and 20 healthy controls (HC), and based on
information collected from the investigated subjects (Appendix I), it was
possible to characterize them demographically in terms of age, duration of
disease, gender, educational status, family history of corresponding illness,
allergy to fish meat, as well as, cigarette smoking and alcohol drinking.
3.1.1 Age
Alzheimer's disease patients had the highest mean of age (76.9 ± 2.9
years) followed by VD patients (72.2 ± 1.7 years). The HC had an age mean
of 66.9 ± 3.1 years, and although it was lower than the observed means in
AD and VD patients, it still represents individuals with old ages. Down's
syndrome patients were presented with the lowest mean of age (15.8 ± 4.9
years), because they were either children or adolescents (Table 3-1)
Table 3-1: Age distribution in Alzheimer's, vascular dementia and Down's
syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Mean ± S.E.
76.9 ± 2.9
72.2 ± 1.7
15.8 ± 4.9
66.9 ± 3.1
Age (Years)
Minimum
38
60
8
44
Maximum
100
92
15
95
It is always augmented that AD and VD are diseases of older ages
(Iqbal et al., 2005); therefore, the present results are in a good agreement
Chapter Three: Results and Discussion
٥۳
================================================================
with such theme, highlighting age (≥ 65 years) as a risk factor for both
morbidities. It has been demonstrated that age-specific prevalence of AD
almost doubles every 5 years after aged 65, and among developed nations,
approximately 1 in 10 older people (≥ 65 years) is affected by some degree
of dementia, whereas more than one third of very old people (≥ 85 years)
may have dementia-related symptoms and signs (Corrada et al., 2008).
However, fewer cases occurred at younger ages, and constituted a portion (46%) of AD. In such cases, family history has been suggested to play an
important role, and early-onset familial AD is often caused by autosomal
dominant mutations in specific genes, especially amyloid precursor protein
(APP), but they account for only 2-5% of all AD patients (Blennow et al.,
2006). For VD, age-related health complications (for instance, diabetes and
cardiovascular diseases) can account for such morbidity in individuals older
than 60 years (Qiu et al., 2007).
3.1.2 Duration of disease
The two groups of dementia (AD and VD) responded oppositely to
duration of disease. In the case of AD, most cases (76.7%) had a duration of
6-15 years, while in VD, 89.3% of patients had a duration of ≤ 5 years. Such
difference was highly significant (P ≤ 0.001), as shown in table( 3-2).
Table 3-2: Alzheimer's and vascular dementia patients distributed by
duration of disease.
Disease Duration
(Years)
≤5
6-15
Alzheimer's Disease
(Number = 30)
No.
%
7
23.3
23
76.7
Vascular Dementia
(Number = 28)
No.
%
25
89.3
3
10.7
Pearson's Chi-square = 25.471; D.F. = 1; P ≤ 0.001 (Significant)
Chapter Three: Results and Discussion
٥٤
================================================================
It has been demonstrated that the typical duration of AD is 8-10 years,
but the course can range from 1 to 25 years, and for unknown reasons, some
AD patients show a steady downhill decline in function, while others have
prolonged plateaus without major deterioration (Roberson and Mucke,
2006). In contrast, VD patients might be at a greater risk of mortality due to
the complications of cardiovascular diseases that may short the duration of
disease (Bennett et al., 2006; Van Oijen , 2007). Such findings fit well what
was observed in the present study, in which AD patients had a much higher
duration of disease than VD patients.
3.1.3 Gender
Although there was no significant difference between AD and VD
distributed by gender, both groups of diseases showed a high frequency of
females than males (66.7 vs. 33.3% in AD and 57.1 vs. 42.9% in VD). The
associated male:female ratios were 1:2 and 1:1.3, respectively, and the
distribution was significantly different (P ≤ 0.05) in AD patients, but not VD
patients (Table 3-3).
Table 3-3: Alzheimer's and vascular dementia patients distributed by gender.
30
Male (M)
No. %
10 33.3
Female(F) M:F
Z
No. % Ratio Value
20 66.7 1:2
2.33
28
12
16
Groups
No.
Alzheimer's
disease
Vascular dementia
42.9
57.1
1:13
0.80
P ≤0.05
0.05
N.S.
Pearson's Chi-square = 0.558; D.F. = 1; P > 0.05 (Not-significant)
These results suggests that females are at greater risk to develop AD
than males, and the same augmentation can be raised for VD, although the
present study evidence is not strongly suggestive. In agreement with such
findings, most studies suggest the female preponderance in AD and VD, and
the risk of development of any form of dementia is reported to be
Chapter Three: Results and Discussion
٥٥
================================================================
approximately twice as high in females as in males and the risk of AD is as
much as three times higher in females (reviewed by Povova et al., 2012).
Such difference can be explained on the basis that AD pathogenesis may be
influenced by metabolic changes induced by sex hormones. Oestrogen is
known to be protective in the brain, and loss of the hormone during
menopause may be responsible for deficits in brain metabolism, which lead
to AD. Male and female brains react very differently to testosterone and
estradiol despite the fact that both sexes have receptors for each hormone
(Ray et al., 2007; Anstey et al., 2008). Both sexes can synthesize estradiol in
neurons, but synaptic response in different brain regions appears to be highly
sexually dimorphic. Therefore understanding AD pathogenesis demands
broad knowledge of the complex interplay among genetic, hormonal, and
environmental influences, and the role of gender differences in the onset and
course of AD remains ill-defined and demands further attention (Phung et
al., 2010).
Gender differences in AD severity have also been found, especially with
regard to dementia and cognition. Cognitive test performance differences
have been documented in healthy men and women, as well as, in patients
with dementia and AD. One study demonstrated relatively equal scores on
naming and fluency tests in patients with dementia, but women with AD had
significantly greater impairment (Yaffe et al., 2003). Another study
compared global cognitive function (last evaluation before death) to specific
measures of plaque and tangle pathology derived from brain autopsy.
Pathology of AD was more likely to manifest as dementia in women than in
men, and for each additional unit of AD pathology, women had a nearly 3fold increase in the odds of having been diagnosed with AD. However,
further studies are needed to correlate diagnosis and AD pathology to
understand why women bear the burden of AD prevalence (Seshadri et al.,
2006; Phung et al., 2010).
Chapter Three: Results and Discussion
٥٦
================================================================
3.1.4 Educational Status
Most of AD patients were illiterate (86.7%), while most of VD patients
had some sort of education (78.6%). Such difference was highly significant
(P ≤ 0.001), and the odds ratio of being illiterate and have AD was 23.83
(Table 3-4).
Table 3-4: Alzheimer's and vascular dementia patients distributed by
educational status.
Groups
Alzheimer's disease
Vascular dementia
No.
30
28
Illiterate
No.
%
26
86.7
6
21.4
Educated
No.
%
4
13.3
22
78.6
Odds ratio= 23.83; Pearson's Chi-square=24.922; D.F.= 1; P ≤ 0.001 (Significant)
Such results clearly suggest that illiteracy is an important risk factor
for AD. In agreement with such scope, most but not all studies have shown
that people with fewer years of education seem to be at higher risk for AD
than those with more years of education (Su et al., 2008). Furthermore, the
combination of low socioeconomic status and elementary school only
education have also been shown to increase the risk of AD threefold
compared to people with high socioeconomic status and higher education
(Blass and Rabins, 2008). This has lead some researchers to believe that a
higher level of education provides a “cognitive reserve” that enables
individuals to better compensate for changes in the brain that could result in
AD or other dementia (Su et al., 2001).
3.1.5 Family History
Six out of 30 AD patients (20.0%) were observed to have a family
history of the disease (father, mother or brother), while the corresponding
frequency in VD patients was higher (32.1%), but the difference was not
Chapter Three: Results and Discussion
٥۷
================================================================
significant. Accordingly, the sporadic cases (especially in AD) were the most
frequent and accounted for 80.0 and 67.9% of AD and VD, respectively
(Table 3-5).
Table 3-5: Alzheimer's and vascular dementia patients distributed by family
history.
Groups
Alzheimer's disease
Vascular dementia
No.
30
28
Family History
Positive
Negative
No.
%
No.
%
6
20.0
24
80.0
9
32.1
19
67.9
Pearson's Chi-square = 0.552; D.F. = 1; P > 0.05 (Not-significant)
As the results suggested, there is no significant difference between AD
and VD with respect to the contribution of family history to both morbidities,
but still there is a proportion of both groups of patients that had a family
history of AD or VD. Actually, studies have demonstrated that the strongest
known risk factor (after APOE ε4 allele) for AD remains a positive family
history, with a three-fold to four-fold higher risk among individuals having a
single first-degree relative with AD and a nearly eight-fold higher risk
among individuals with two or more first-degree relatives with AD (Lott et
al., 2006). However, the majority of AD cases are sporadic (an observation
that is strengthen by the current results), and present considerable
heterogeneity in terms of risk factor profiles and neuropathological features
(Green et al., 2002), and although first-degree relatives of Alzheimer's
patients have a higher lifetime risk of developing AD than the general
population or relatives of non-demented individuals; both genetic and shared
environmental factors contribute to the phenomenon of familial aggregation
(Huang et al., 2004). Furthermore, it has also been demonstrated that
sporadic late-onset AD accounts for the majority of all AD cases, and this
form can likely be caused by a number of gene mutations, combined with
Chapter Three: Results and Discussion
٥۸
================================================================
aging and exposure to environmental agents. The most well-established
genetic risk factor for development of sporadic late-onset AD is inheritance
of the ε4 allele of APOE gene (Goedert and Spillantini, 2006).
3.1.6 Allergy to Fish Meat
Allergy to fish meat was observed in 33.3% of AD patients, while it was
17.9% in VD patients, but the difference did not reach a significant level
(Table 3-6).
Table 3-6: Alzheimer's and vascular dementia patients distributed by allergy
to fish meat.
Groups
Alzheimer's disease
Vascular dementia
No.
30
28
Allergy to Fish Meat
Allergic
Not Allergic
No.
%
No.
%
10
33.3
20
66.7
5
17.9
23
82.1
Pearson's Chi-square = 1.809; D.F. = 1; P > 0.05 (Not-significant)
Although the difference was not significant, we still have one third of
AD patients who were allergic to fish meat. Such observation may question
fish meat allergy as a risk factor for AD. It is difficult to explain that, but
such nutritional status may consequence in depriving the patients from some
necessary fish meat-related nutritional constituents; for instance omega-3
fatty acid. In an epidemiological study, it was found that if two dietary habits
(for instance, omega-3 fatty acid and fruit/vegetable consumption) are
present, the risk for dementia especially AD was significantly reduced
(Ngandu et al., 2007). Such observation is supported by experimental
evidence, in which it has been demonstrated that administering DHA (long
chain omega-3 fatty acid) to aged Alzheimer's-prone rats reduced total
amyloid-beta by more than 70% compared with low-DHA or control chow
diets, and image analysis of brain sections showed that plaque burden was
Chapter Three: Results and Discussion
٥۹
================================================================
reduced by more than 40% (Lim etal.,2005). Furthermore, large body of
human epidemiological studies indicated that dietary fish consumption
reduces the risk of AD (Beydoun et al., 2007).
3.1.7 Cigarette Smoking
Smoker and non-smoker AD patients were observed with a similar
frequency (50%), while in VD patients, smokers were higher than nonsmokers (53.6 vs. 46.4%), but the difference was not significant ( Table 3-7).
Table 3-7: Alzheimer's and vascular dementia patients distributed by
cigarette smoking.
Groups
Alzheimer's disease
Vascular dementia
No.
30
28
Cigarette Smoking
Smoker
Non-smoker
No.
%
No.
%
15
50.0
15
50.0
15
53.6
13
46.44
Pearson's Chi-square = 0.047; D.F. = 1; P > 0.05 (Not-significant)
The present results are not in favour of that cigarette smoking is a risk
factor for AD or VD, although other investigations have suggested that
cigarette smoking is either protective or can increase the risk to develop AD
(Tyas et al., 2003; Aggarwal et al., 2006; Anstey et al., 2007; Peters et al.,
2008). This result may be because the small number of study samples or
because the women ratio was higher than men, so the most women not
smoking.
3.1.8 Alcohol Drinking
Most of AD and VD patients were not alcoholic (80.0 and 85.7%,
respectively) (Table 3-8), and accordingly it is not possible to consider
alcohol drinking is a risk factor for AD or VD in the present samples of
patients, as suggested by other investigators (Anttila et al., 2004; Ding et al.,
Chapter Three: Results and Discussion
٦۰
================================================================
2004; Paul et al., 2008). Such difference is probably related to the fact that
we do not have the right measure of registry of alcoholism in the patients,
and the only source of information is either the patient or the individual who
accompanied it and the most study samples were women, so most women
not drinking.
Table 3-8: Alzheimer's and vascular dementia patients distributed by alcohol
driniking.
Groups
No.
Alzheimer's disease
Vascular dementia
30
28
Alcohol Drinking
Alcoholic
Not Alcoholic
No.
%
No.
%
6
20.0
24
80.0
4
14.3
24
85.7
Pearson's Chi-square = 0.331; D.F. = 1; P > 0.05 (Not-significant)
3.2 Serum Level of Beta Amyloid (Aβ)
The highest level of Aβ was observed in AD patients (56.81 ± 4.19
pg/ml), followed by DS (34.20 ± 4.77 pg/ml) and VD (23.8 ± 1.64 pg/ml)
patients, while controls were presented with the lowest mean (9.87 ± 1.05
pg/ml). However, the means of the four groups were significantly (P ≤ 0.05)
different (Table 3-9).
Distributing AD and VD patients by gender revealed that females had a
significantly higher serum level of Aβ than males of both groups of patients
(AD: 62.44 ± 5.5 vs. 46.57 ± 5.35 pg/ml; VD: 26.2 ± 2.3 vs. 20.6 ± 1.8
pg/ml), as shown in Table (3-10).
Table 3-9: Serum level of beta amyloid in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Groups
Alzheimer's disease
No.
30
Serum level of Beta Amyloid (pg/ml)
Mean ± SE*
Minimum
Maximum
56.81 ± 4.19A
17.8
125
Chapter Three: Results and Discussion
٦۱
================================================================
Vascular dementia
Down's syndrome
Controls
28
10
20
23.8 ± 1.64C
34.20 ± 4.77B
9.87 ± 1.05D
12.4
14
4.5
42
55
21.2
*Different letters: Significant difference (P ≤ 0.05) between means.
Table 3-10: Serum level of beta amyloid in Alzheimer's and vascular
dementia distributed by gender.
Groups
Alzheimer's disease
Vascular dementia
Gender
No.
Male
Female
Male
Female
Serum level of Beta Amyloid (pg/ml )
Mean ± SE*
Min.
Max.
10
20
12
46.57 ± 5.35B
62.44 ± 5.5A
20.6 ± 1.8B
18.9
17.8
12.4
67
125
32.4
16
26.2 ± 2.3A
13.9
42.2
*Different letters: Significant difference (P ≤ 0.05) between means.
These results clearly suggest the role of Aβ in aetiopathogenesis of
dementia of AD, DS and VD patients, with a major contribution in AD
patients (5.8 times of the control value), followed by DS (3.5 times) and
finally VD (2.4 times); an observation that shares the interest of other
investigators in the field of dementia that is associated with different
pathologies (Mehta et al., 2000;Joseph et al., 2008).
Beta amyloid is a normal product of APP processing (Estus et al., 1992)
and is a normal soluble component of the plasma and the cerebrospinal fluid
(Seubert et al., 1992). The observation of Aβ deposits in the senile plaques
(SPs) in essentially all cases of AD has led to the hypothesis that a
conversion of soluble Aβ into insoluble fibrils is critical for the onset of the
disease. This hypothesis is supported by the fact that fresh Aβ is non-toxic to
cultured neurons, while aged Aβ (incubated to form amyloid fibrils) becomes
toxic (Howlett et al., 1995).
It has also been suggested that Aβ might be associated with free radical
formation, but the mechanism behinds this has not been fully understood.
Chapter Three: Results and Discussion
۲۲
================================================================
The full-length Aβ peptides possess a Cu+2 binding domain and Aβ1–42 can
reduce the bound Cu+2 to Cu+ (Atwood et al., 2004). The resultant Aβ1–42
associated Cu+ was reported to lead to H2O2 production. This transfer of a
single electron from the peptide to the metal would result in the formation of
a peptidyl free radical, which is one possible explanation for the formation of
Aβ associated free radicals (Guilloreau et al., 2007).
Furthermore, amino acid sequence analysis revealed that Aβ1–42 peptide
contains a single methionine at residue 35 (Met35), which has been
demonstrated to be susceptible to oxidation in vivo, especially under
conditions of oxidative stress; therefore a number of studies have focused on
the role of Met35 in AD (Pogocki and Schӧneich, 2002; Schӧneich et al.,
2003; Butterifeld and�Boyd-Kimball, 2005). An examination of senile
plaque-resident A. 1�42 showed a high proportion of methionine sulfoxide,
and accordingly, it has been suggested to participate in free radical reaction
and formation (Butterifeld and Bo��-Kimball, 2005). Structural studies also
revealed that oxidation of methionine residues in model peptides is
significantly alter the secondary structure of �β1�42, and methionine
oxidation to sulfoxide leads to predominantly . -sheet conformation, which is
the conformation adopted by toxic �. (Labrenz et al., 2008).
Beta amyloid was also observed with a significant increased level in
sera of DS patients, and this can be explained by the fact that APP is coded
for by gene on chromosome 21, which is involved in DS trisomy, and
persons with DS have been reported to have increased Aβ deposits (they
have extra copy of chromosome 21) and can eventually develop AD (Estus
et al., 1992). Such observation further confirms the role of Aβ in AD, and
leads to the accumulation of Aβ in the brain; therefore an initiation of
inflammation in AD brain can result in activation of microglia and release of
neurotoxic substances, and these processes lead to neuronal degeneration
(Bing-Tian et al., 2012). Beta amyloid may also induce oxidative stress by
Chapter Three: Results and Discussion
۳۳
================================================================
causing mitochondrial dysfunction (after entering the mitochondria), which
results in an increase of ROS and a decrease in the level of endogenous
antioxidant such as glutathione peroxidase, super oxide dismutase and
catalase. Moreover, Aβ can induce nitric oxide generation by up regulating
of expression of nitric oxide synthase which plays a pivotal role in the
cascade of events that lead to neuronal death (Wanga et al., 2012).
3.3 Lipid Profile
A. Total Cholesterol
The highest serum level of total cholesterol was observed in VD
patients (264 ± 15 mg/dL), which represented a significant (P ≤ 0.01)
difference in comparison with AD, DS and controls (161 ± 7, 214 ± 9 and
184 ± 10 mg/dL, respectively). In DS patients, it was also increased, but the
difference was significant in comparison with AD patients only. In AD
patients, the lowest level of total cholesterol was observed, but it was not
significant as compared with controls (Table 3-11).
Table 3-11: Serum level of total cholesterol in Alzheimer's, vascular
dementia and Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of Total Cholesterol (mg/dL)
Mean ± SE*
Minimum
Maximum
C
161 ± 7
107
250
264 ± 15A
156
465
214 ± 9B
145
250
184 ± 10BC
134
300
*Different letters: Significant difference (P ≤ 0.05) between means.
B. Triglycerides
The serum level means of triglycerides in AD and VD patients were
(203 ±15 and 189±11 mg/dL), respectively, which were not significantly
different, but they were significantly higher than the corresponding means in
Chapter Three: Results and Discussion
٤٤
================================================================
DS and controls (142 ±5 and 123 ± 11 mg/dL), respectively. However, the
latter two means were not significantly different (Table 3-12).
Table 3-12: Serum level of triglycerides in Alzheimer's, vascular dementia
and Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of Triglycerides (mg/dL)
Mean ± SE*
Minimum
Maximum
203 ± 15A
110
453
189 ± 11A
100
300
142 ± 5B
123
170
B
123 ± 11
34
254
*Different letters: Significant difference (P ≤ 0.05) between means.
C. High Density Lipoproteins (HDL) Cholesterol
There was a significantly decreased serum level of HDL cholesterol in
AD, VD and DS patients (33.4 ± 1.2, 41.5 ± 1.8 and 38.3 ± 1.7 mg/dL,
respectively) as compared with control group (57.4 ± 2.7 mg/dL). The lowest
level was in AD, and it was significant as compared with VD patients, but
not with DS patients (Table 3-13).
Table 3-13: Serum level of high density lipoproteins cholesterol in
Alzheimer's, vascular dementia and Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of High Density Lipoproteins
Cholesterol (mg/dL)
Mean ± SE*
Minimum
Maximum
33.4 ± 1.2C
16
42
B
41.5 ± 1.8
27
58
BC
38.3 ± 1.7
30
48
A
57.4 ± 2.7
30
81
*Different letters: Significant difference (P ≤ 0.05) between means.
D. Low Density Lipoproteins (LDL) Cholesterol
Chapter Three: Results and Discussion
٦٥
================================================================
The highest mean level of LDL cholesterol was observed in VD patients
(185.0 ± 15.2 mg/dL), and the difference was significant in comparison with
AD and DS patients and controls (84.5 ± 7.7, 146.0 ± 10.0 and 97.7 ± 10.9
mg/dL, respectively). After VD, DS patients were observed to have a
significant increase of LDL cholesterol as compared with AD patients or
controls. However, AD patients and controls demonstrated no significant
difference between their means (Table 3-14).
Table 3-14: Serum level of low density lipoproteins cholesterol in
Alzheimer's, vascular dementia and Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of Low Density Lipoproteins
Cholesterol (mg/dL)
Mean ± SE*
Minimum
Maximum
C
84.5 ± 7.7
18
200
A
185.0 ± 15.2
76
400
146.0 ± 10.0B
78
181
97.7 ± 10.9C
19
218
*Different letters: Significant difference (P ≤ 0.05) between means.
E. Very Low Density Lipoproteins (VLDL) Cholesterol
The mean serum level of VLDL cholesterols showed no significant
difference between AD and VD patients (40.7 ± 2.9 and 37.9 ± 2.1 mg/dL,
respectively), but both means were significantly higher than the means of DS
patients and controls (28.3 ± 0.9 and 24.6 ± 2.1 mg/dL, respectively).
However, the latter two means were significantly not different (Table 3-15).
Table 3-15: Serum level of very low density lipoproteins cholesterol in
Alzheimer's, vascular dementia and Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
No.
30
28
Serum Level of Very Low Density
Lipoproteins Cholesterol (mg/dL)
Mean ± SE*
Minimum
Maximum
40.7 ± 2.9A
22
91
37.9 ± 2.1A
20
60
Chapter Three: Results and Discussion
٦٦
================================================================
Down's syndrome
Controls
10
20
28.3 ± 0.9B
24.6 ± 2.1B
25
7
34
51
*Different letters: Significant difference (P ≤ 0.05) between means.
Most of the investigated lipid profile parameters were presented with
different means in the four studied groups (AD, VD, DS and controls), and
each group was characterized with a lipid parameter either in term of
increased or decreased mean. To shed light on such characterization, each
lipid parameter was presented as a percentage of the total sum of its serum
level in the four investigated groups (Figures 3-1, 3-2, 3-3, 3-4 and 3-5). For
AD, the highest percentage was observed in triglycerides (30.9%) and VLDL
cholesterol (31.0%) (Figures 3-2 and 3-5). In VD, total cholesterol was
observed with the highest percentage, which was 32.1% (Figure 3-1). It was
also interesting to note that controls scored the highest percentage of HDL
cholesterol (33.6%), as shown in figure (3-2).
Figure 3-1: Total cholesterol
percentage of the total sum of
its serum level in Alzheimer's,
vascular dementia and Down's
syndrome
patients
and
controls.
Chapter Three: Results and Discussion
۷۷
================================================================
Figure 3-2: Triglycerides
percentage of the total sum
of its serum level in
Alzheimer's,
vascular
dementia
and
Down's
syndrome patients and
controls.
Figure 3-3: High density
lipoproteins cholesterol
percentage of the total
sum of its serum level in
Alzheimer's,
vascular
dementia and Down's
syndrome patients and
controls.
Figure 3-4: Low density
lipoproteins cholesterol
percentage of the total
sum of its serum level in
Alzheimer's,
vascular
dementia and Down's
syndrome patients and
controls.
Figure 3-5: Very low
density
lipoproteins
cholesterol percentage of
the total sum of its serum
level in Alzheimer's,
vascular dementia and
Down's
syndrome
patients and controls.
Chapter Three: Results and Discussion
۸۸
================================================================
It is obvious from these results that the serum level of total cholesterol
was decreased in AD patients, and although it was within the normal range,
other studies demonstrated similar findings (Giovanni et al., 2006; Reitz et
al., 2010). However, the studies also agree that cholesterol might be involved
in the pathogenesis of VD (Selkoe, 2002); an observation that is depicted in
table (3-11) and figure (3-1), in which VD patients dominated the serum
level of total cholesterol.
Plasma lipids are transported in blood as lipoproteins, which are
composed of apolepoprotein (APOE) plus plasma lipids (Tang, 2009).
Apolepoprotein has the capacity to render lipids compatible with the aqueous
environment of body fluids and enables their transport throughout the body
to tissues where they are required and facilitates the transport of cholesterol
from blood to all cells via specific receptor-based mechanisms in plasma
membranes (Perdomo and Dong, 2009). This APOE is synthesized in
astrocytes, which are specialized glial cells found in the brain, and therefore
its role in the pathogenesis of AD can not be ignored (Tang, 2009). In the
present AD patients, the low serum level of total cholesterol may reflect a
malnutrition in the patients, because most of AD patients experience
anorexia (loss of appetite), otherwise, a defect in cholesterol metabolism
could have occurred. In addition, lipid serum levels have been observed to be
Chapter Three: Results and Discussion
۹۹
================================================================
decreased with aging, but may not have the same significance they have in
middle age (Maulik et al., 2013).
It has also been demonstrated that high cholesterol level (within the
normal range) may enhance a protective mechanism against AD progression,
by altering the degradation of APP and then affecting the Aβ role in the
pathogenesis of the disease (Fratiglioni et al., 2008) Furthermore, APOE in
astrocytes has been shown to be able to control the level of Aβ in neurons,
by inducing an increase or a decrease in its concentration; depending upon
the status of lipids in the cell (Poirier, 2003). In addition, APOE enhances
Aβ clearance when sufficient fatty acids are present, but increases Aβ
concentration when it is unattached with lipid (Maulik et al., 2013).
High cholesterol level (within the normal range) has also been
positively correlated with longevity in people over 85 years old, and in some
cases has been shown to be associated with a better memory function and a
reduced dementia (Michikawa, 2004). Also, cholesterol cause the
lipoproteins in the cell membrane (which is responsible to transport plasma
lipid by attaching with it) to pack into a tighter molecular configuration, such
as the fatty acids are protected from exposure to oxidative damage
(Michikawa, 2006). Furthermore, it has been demonstrated that neurons in
the AD brain are chronically exposed to excessive amount of glutamate, as
well as hydrogen peroxide and hydroxyl radicals due to mitochondrial
defects, and then with insufficient fatty acid supply to repair cell membrane
damage, the cells may undergo apoptosis (Vance, 2006).
With respect to HDL cholesterol, the three groups of patients (AD, VD
and DS) shared a significant decreased level of HDL cholesterol as
compared with healthy controls, and the highest decrease was observed in
AD patients; an observation that may implicate the protective role of HDL
cholesterol against AD development. Such implication has been recently
strengthen in a study carried out by Reitz et al. (2010), in which the authors
Chapter Three: Results and Discussion
۷۰
================================================================
demonstrated that higher levels of HDL cholesterol were associated with a
decreased risk to develop AD or dementia, and further confirmation has also
been presented by Reitz et al. (2010), who demonstrated that high HDL
cholesterol levels in elderly individuals may be associated with a decreased
risk of AD. It has also been demonstrated that low HDL cholesterol level is a
risk factor for cerebrovascular disease, and treatment with lipid-lowering
medications can prevent stroke. Stroke is associated with higher AD risk,
and may interact with amyloid pathology in an additive way and lower the
amyloid burden necessary to precipitate dementia (Sacco et al., 2001).
Furthermore, low concentrations of HDL cholesterol are known to be
independent risk factors for carotid artery atherosclerosis, which in turn may
lead to cognitive impairment through cerebral hypoperfusion, embolism, or
disruption of white matter (Mooradian, 2009). High-density lipoprotein
cholesterol might also be linked with small-vessel disease by playing a role
in the removal of excess cholesterol from the brain by interaction with
APOE and heparan sulfate proteoglycans in the subendothelian space of
cerebral microvessels. Thus, a low HDL cholesterol level could precipitate
AD through a cerebrovascular pathway (Reitz et al., 2010).
The other lipid parameter that showed a significant increased level in
AD patients was triglycerides; an observation that may suggest their
potential as a risk factor for AD, or their homeostasis is modified in the
disease. It has been demonstrated that plasma triglycerides homeostasis
depends on the balance between triglycerides secretion and lipolysis, and
such balance has been found to be affected by plasma Aβ, in which APOE
may play a role. Beta amyloid can bind APOE in the brain, and Aβ has been
reported to be associated with APOE-containing lipoproteins in plasma, and
APOE-deficient mice displayed impaired triglycerides secretion and
adenoviral delivery of exogenous APOE resulted in a dose-dependent
increase in triglycerides secretion (Wilson et al., 2006). Other work has
Chapter Three: Results and Discussion
۷۱
================================================================
shown that radiolabeled exogenous Aβ can associate with synthetic
chylomicron-like lipid emulsions and is metabolized in parallel with these
particles after injection into the peripheral circulation of rabbits (James et al.,
2003). These results, along with our observation of increased triglycerides
level in AD patients, suggest a model whereby hepatic uptake of Aβlipoprotein complexes could result in enhanced secretion of VLDL particles,
which were also increased in the present AD patients; thereby elevating
plasma triglycerides levels might have occurred prior to amyloid deposition,
as suggested by Burgess et al. (2006). However, additional studies will be
necessary to determine which receptors mediate uptake of endogenously
produced and transported Aβ, and to elucidate the mechanisms underlying
enhanced triglycerides and VLDL secretion in vivo.
The presented results and discussion strongly suggest that some plasma
lipid parameters are involved in the aetiopathogenetic mechanism of AD,
especially if the results are interpreted in the context of Aβ, and manipulating
the plasma level of these lipid parameters may have therapeutic potential.
Actually, a considerable interest has been promoted in determining whether
pharmacological manipulation of lipid levels may provide therapeutic benefit
for AD. Initial retrospective studies suggested that the prevalence of AD
could be reduced by up to 70% by statins, drugs that inhibit HMG Co-A
reductase, which catalyses the rate-limiting step in cholesterol biosynthesis
(Buxbaum et al., 2002). Furthermore, inhibition of cholesterol biosynthesis
by statins or other compounds decreases amyloid burden in guinea pigs and
in transgenic murine models of AD (Vega et al., 2003), and in humans, statin
treatment reduced the levels of 24hydroxycholesterol, the major cholesterol
metabolite of the brain (Hoglund et al., 2005). However, the efficacy of
statins to affect Aβ levels, the prevalence or incidence of AD, or cognitive
function remains to be fully elucidated, although several studies suggest that
Chapter Three: Results and Discussion
۲۲
================================================================
statins may have a neuroprotective effect especially in subjects with mild AD
(Pandey et al., 2013).
3.4 Total Antioxidant Capacity (TAC)
The lowest TAC was observed in AD patients (5.29 ± 0.46 nmol/μL) as
compared with VD patients (8.85 ± 0.40 nmol/μL), DS patients (7.24 ± 1.07
nmol/μL) or controls (9.65 ± 0.67 nmol/μL), but the difference attended a
significant level (P ≤ 0.05) in comparison with VD patients and controls.
Down's syndrome patients also showed a significant decreased TAC in
comparison with controls (Table 3-16). Distributing these groups according
to gender revealed no significant difference between males and females of
each group.
Table 3-16: Serum level of total antioxidant capacity in Alzheimer's,
vascular dementia and Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of Total Antioxidant Capacity
(nmol/μL)
Mean ± SE*
Minimum
Maximum
C
5.29 ± 0.46
2
12
8.85 ± 0.40AB
4
13
7.24 ± 1.07BC
3.6
12
9.65 ± 0.67A
6
16
*Different letters: Significant difference (P ≤ 0.05) between means.
The main observation in the present study was a decreased TAC in AD
patients by almost 50.0% of the control value, with a less involvement in DS
patients (25.0%), and this may highlight the role of TAC in pathogenesis of
AD. In this regard, some investigations agree that TAC is reduced in sera or
plasma of AD patients (Pulido et al., 2005), while others reported no change
in TAC between AD patients and controls (Kusano and Ferrari, 2008).
However, it has been demonstrated that aerobic cells generate reactive
Chapter Three: Results and Discussion
۳۳
================================================================
oxidative species, particularly in the oxidation–reduction reactions necessary
for the generation of ATP, and in specialized cells with high metabolic
activity, such as neurons, the number of free radicals produced is estimated
about 1011 reactive oxidative species/cell/day (Petersen et al., 2007).
Furthermore, it has been suggested that the human brain is particularly
vulnerable to oxidative stress as a result of the relatively low levels of
antioxidants, high levels of polyunsaturated fatty acids and increased need of
oxygen (Sultana et al., 2008). In addition, there are many evidences that
suggest that oxidative stress is one of the earliest events in AD pathogenesis
and plays a key role in the development of the AD pathology (Zhu et al.,
2004; 2007; Bonda et al., 2010), and an accumulation of products of free
radicals damaging central nervous system (CNS) in subjects with AD has
also been described (Butterifeld�et al., 2007; Mangialasche et al., 2009).
Accordingly, it is believed that oxidative damage to critical molecules occurs
early in the pathogenesis of AD; perhaps is the earliest feature of an AD
brain and precedes pronounced neuropathological alterations (Baldeiras et
al., 2008). In fact, some evidences have suggested that the Aß deposition in
AD neurons may be considered as an effort to protect these cells against
damage due to oxidative stress (Hayashi et al., 2007; Nakamura et al., 2007).
In agreement with such scope, and as demonstrated in the present study, it is
possible to suggest that a reduction of TAC defenses may render body cells
and tissues to become more prone to develop dysfunction and/or disease; and
then, the maintenance of adequate antioxidant levels, but not overdosage, is
essential to prevent or even manage a great number of disease conditions.
Accordingly, it has been strongly described to consider the use of TAC as a
biomarker of disease in biochemistry, medicine, food and nutritional
sciences, and in many different pathophysiological conditions (heart and
vascular diseases, diabetes mellitus, neurological and psychiatric disorders
including AD, renal disorders and lung diseases), TAC could be a reliable
Chapter Three: Results and Discussion
٤٤
================================================================
biomarker of a diagnostic value (Kusano and Ferrari, 2008). Such suggestion
has been recently strengthened by two recent studies, in which, oxidative
damage could be one important aspect for the onset of AD, and oxidative
stress markers could be useful to diagnose the illness in their earliest stages
(Puertas et al., 2012; Skoumalová and Hort, 2012).
3.5 C-reactive Protein (CRP)
A common theme between AD and VD patients was presented by a
significant increased serum level CRP (5.17 ± 0.52 and 4.39 ± 0.48 mg/L,
respectively) in comparison with DS patients (2.19 ± 0.16 mg/L) or controls
(1.79 ± 0.21 mg/L) (Table 3-17).
Table 3-17: Serum level of C-reactive protein in Alzheimer's, vascular
dementia and Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of C-reactive Protein (mg/L)
Mean ± SE*
Minimum
Maximum
A
5.17 ± 0.52
1
10
4.39 ± 0.48A
1
10
2.19 ± 0.16B
1
3
1.79 ± 0.21B
1
4
*Different letters: Significant difference (P ≤ 0.05) between means.
It is almost agreed by most other studies that CRP shows an increased
serum level in AD and VD patients (Joseph et al., 2008; Lepara et al., 2009;
Thambisetty and Lovestone, 2010; Bing-Tian et al., 2012), in which a CRP
serum level is greater than CRP >3 mg/L has been associated with impaired
cognition and increase risk of VD and AD. Furthermore, Dlugai et al. (2012)
reported that elevated serum levels of CRP level are associated with at least
twofold increased probability of cognitive impairment. Accordingly, an
elevated serum level of CRP might be a useful biomarker, especially in
Chapter Three: Results and Discussion
۷٥
================================================================
association with TAC and Aβ, to identify individuals who are at an increased
risk for memory impairment and dementia (Roberts et al., 2009).
C-reactive protein is not only synthesized by hepatocytes but also by
other cell types, such as neurons; therefore its elevation in serum, CSF and
brain tissue of AD patients might be expected to be due to inflammatory
reactions (Mancinella et al., 2009; Roberts et al., 2009), and although the
exact causes of AD remain elusive, theories continue to support the
involvement of inflammation in AD development (Town et al., 2005).
Evidence on the involvement of inflammation in AD pathogenesis has been
increasingly documented, and as CRP is an acute-phase protein during
inflammation, it may play a central role in the immune response of the brain.
This immune response, while it is designed to protect the host, can be also
destructive to host tissue when misdirected, and studies indicated that CRP
can stimulate autodestruction and enhance phagocytosis in the progression of
AD (Salminen et al., 2009), and can also independently induce AD-like
cognitive impairment (Lim et al., 2005). Therefore, the association of CRP
with AD pathogenesis can not be ignored, especially if it is considered in
association with aberrant Aβ, which is a hallmark of AD pathology.
With respect to VD, most cases have been presented with a chronic
inflammatory process, and it has been found that inflammation increases the
risk to develop cardiovascular diseases, which in turn might be associated
with impaired cognition (Petersen et al., 2007). However, an adjustment for
several vascular factors has revealed that the association between CRP and
impaired cognition might be slightly modified, indicating that the association
between high CRP and poor memory is not total mediated through vascular
factors. Thus, inflammatory markers such as CRP may provide an additional
method for global assessment of cardiovascular risk and cognitive
impairment (Teunissen and Scheltens, 2007).
Because, inflammatory
mechanism have been suggested to be involved in cognitive impairment and
Chapter Three: Results and Discussion
٦٦
================================================================
dementia, and CRP have been found in and around Aβ plaques in the brains
of patients with dementia. Accordingly, Tan et al., (2007) suggested that
CRP is a sensitive marker of systemic low-grad inflammation and increased
serum level of CRP can be associated with impaired cognition and an
increased risk of VD and AD.
3.6 Alpha 1-antitrypsin (α1-antitrypsin)
Serum level of α1-antitrypsin was significantly increased in AD and
DS patients (275 ± 23 and 238 ± 10 mg/dL, respectively) in comparison with
controls (181 ± 9 mg/dL), while VD patients (220 ± 58 mg/dL) did not show
any significant difference as compared with AD and DS patients or controls
(Table 3-18).
Table 3-18: Serum level of alpha 1-antitrypsin in Alzheimer's, vascular
dementia and Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of alpha 1-antitrypsin Protein
(mg/dL)
Mean ± SE*
Minimum
Maximum
A
275 ± 23
87
515.5
220 ± 58AB
100
1765
238 ± 10A
198
293
181 ± 9B
120
266
*Different letters: Significant difference (P ≤ 0.05) between means.
There is no direct evidence to support the present findings, but Maes et
al. (2006) demonstrated that α1-antitrypsin levels were significantly
increased in plasma of AD patients, and such elevation was correlated with a
heme
oxygenase-1
suppressor
(HOS)
activity
and
α1-antitrypsin
immunodepletion attenuated HOS activity of AD plasma, and accordingly
the author suggested that α1-antitrypsin may curtail Heme oxygenase-1 (HO1)-dependent derangement of cerebral iron homeostasis and account for
Chapter Three: Results and Discussion
۷۷
================================================================
diminished HO-1 expression in AD peripheral tissues. In a more recent
investigation, the genetic polymorphism of α1-antitrypsin was investigated
in AD patients, and the genetic variant M was reported to be the more
prevalent (Marklová et al., 2012). Furthermore, a link between
polymorphism and increased vulnerability to white matter brain disease in
elders has been described for ‘silent’ heterozygous S and Z α1-antitrypsin
carriers (Schmechel, 2007). These demonstrations together with the findings
of present study, may suggest a potential for α1-antitrypsin in the
aetiopathogenesis of AD.
3.7 Immunoglobulins A, G and M
A. Immunoglobulin A (IgA): Down's syndrome patients demonstrated
the highest serum level of IgA (482 ± 30 mg/dL), which was significantly
higher than the recorded level in VD patients (348 ± 35 mg/dL) and controls
(296 ± 38 mg/dL), but not AD (397 ± 32 mg/dL). The latter three groups
showed no significant difference between their means of IgA (Table 3-19)
Table 3-19: Serum level of IgA in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of IgA (mg/dL)
Mean ± SE*
Minimum
Maximum
AB
397 ± 32
157
903
348 ± 35B
78
756
482 ± 30A
347
605
296 ± 38B
55
589
*Different letters: Significant difference (P ≤ 0.05) between means.
B. Immunoglobulin G (IgG): There was no significant difference
between the means of IgG in AD and VD patients (1246 ± 118 and 996 ±
131 mg/dL, respectively), but the mean of AD was significantly higher than
Chapter Three: Results and Discussion
۸۸
================================================================
the mean of DS patients (708 ± 130 mg/dL) or controls (584 ± 84 mg/dL)
(Table 3-20).
Table 3-20: Serum level of IgG in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of IgG (mg/dL)
Mean ± SE*
Minimum
Maximum
1246 ± 118A
108
2286
996 ± 131AB
321
2286
708 ± 130BC
126
1192
C
584 ± 84
126
973
*Different letters: Significant difference (P ≤ 0.05) between means.
C. Immunoglobulin M (IgM): The four investigated groups (AD, VD,
DS and controls) demonstrated an approximated mean of serum IgM level,
and there was no significant difference between them (Table 3-21).
Table 3-21: Serum level of IgM in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of IgM (mg/dL)
Mean ± SE*
Minimum
Maximum
A
220 ± 21
72
466
A
246 ± 13
109
370
A
194 ± 16
125
291
A
197 ± 14
104
338
*Similar letters: No significant difference (P > 0.05) between means.
The
presented
results
may
not
qualify
the
three
assessed
immunoglobulins (IgA, IgG and IgM) as markers for AD, VD or DS, with
the exception of IgG in AD patients, which showed a significant increased
level, and IgA in DS patients, which also showed a significant increased
level. There is no plausible explanation for the increase of IgG in AD
patients, but it may reflect a state of chronic infection. In this context, it has
been hypothesized that infection with several important pathogens could
Chapter Three: Results and Discussion
۹۹
================================================================
constitute risk factors for cognitive impairment, dementia, and AD in
particular. The authors also summarized the data related to infectious agents
that appear to have a relationship with AD. Infections with herpes simplex
virus type 1, picornavirus, Borna disease virus, Chlamydia pneumoniae,
Helicobacter pylori, and spirochete were reported to contribute to the
pathophysiology of AD or to cognitive changes, and based on these reports,
it may be hypothesized that central nervous system or systemic infections
may contribute to the pathogenesis or pathophysiology of AD, and chronic
infection with several pathogens should be considered a risk factor for
sporadic AD (Honjo et al., 2009).
In the case of DS, there is no direct evidence to support or contradict
the current increased serum level of IgA. However, as IgA is the
immunoglobulin that is associated with mucosal immunity, and as coeliac
disease (CD) is normally associated with an increased serum level of antitissue transglutaminase IgA antibody, a co-existence of DS and CD have
been occasionally reported, but a clear relationship between them has not
been definitely established (Cogulu et al., 2003; Bonamico, 2005). In a more
recent investigation, it has been suggested the need for systematic screening
for CD in children with DS because symptoms that are characteristic of both
diseases may overlap, and their suggestion was based on the findings of
different serum IgA antibodies (Pavlović et al., 2012). Unfortunately, the
present DS patients were not clinically evaluated for CD.
3.8 Third and Fourth Components of Complement
A. Third Component of Complement (C3): Serum level of C3 was
exceptionally and significantly increased in AD patients (179 ± 10 mg/dL),
as compared with VD and DS patients (135 ± 9 and 114 ± 8 mg/dL,
respectively) or controls (134 ± 8 mg/dL) (Table 3-22).
Chapter Three: Results and Discussion
۸۰
================================================================
Table 3-22: Serum level of C3 in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
No.
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
30
28
10
20
Serum Level of C3 (mg/dL)
Mean ± SE* Minimum
Maximum
179 ± 10A
61
312
135 ± 9B
67
263
B
114 ± 8
71
159
B
134 ± 8
64
208
*Different letters: Significant difference (P ≤ 0.05) between means.
B. Fourth Component of Complement (C4): The highest serum
level of C4 was observed in AD patients (51.5 ± 2.7 mg/dL), but the
difference reached a significant level in comparison with DS patients (37.9 ±
3.6 mg/dL), but not VD patients (49.4 ± 6.0 mg/dL) or controls (46.4 ± 5.7
mg/dL) (Table 3-23).
Table 3-23: Serum level of C4 in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of C4 (mg/dL)
Mean ± SE*
Minimum
Maximum
A
51.5 ± 2.7
16
86
AB
49.4 ± 6.0
7.4
118
B
37.9 ± 3.6
22.7
56.8
AB
46.4 ± 5.7
7.4
107
*Different letters: Significant difference (P ≤ 0.05) between means.
It is generally agreed that the complement components are activated in
AD patients (Stoltzner et al. 2000), and in the present study, C3 was
exceptionally increased in AD patients, followed by C4, although the latter
represented a non-significant difference. Such activation is though to be
trigged in AD brain primarily by the interaction of complement proteins with
the aggregated forms of Aβ, and soluble non-fibrillar Aβ may also be
capable of activating complement (Roberts et al., 2009). Complement
Chapter Three: Results and Discussion
۸۱
================================================================
activation and plaque formation are mutually promoting mechanisms, and it
has been demonstrated that aggregated Aβ efficiently binds C1q, which in
turn activates the classical complement pathway, and this process further
enhances Aβ aggregation and fibril formation (David et al., 2008). However,
complement activation in AD was initially reported to be limited to the
classical pathway, but the alternative pathway activation was also reported,
and it is well-known fact that C3 is involved in both pathways of
complement activation, and in vitro, Aβ fibrils (fAβ) have been shown to
activate both the classical complement pathway by directly binding to C1q
and the alternative pathway via interactions with C3 (Zhou et al., 2008).
It has been suggested that complement can enhance neurotoxic effects
in AD brain by increasing Aβ aggregation and such process potentiates the
neurotoxicity and can attract microglia and promotes their secretion of
inflammatory cytokines, which may contribute further to the neurodegenerative process in AD (Broughton et al., 2012). Activation of locally
produced complement factors may also act as a mediator between amyloid
deposits and neurodegenerative changes seen in AD, and accordingly,
complement activation products have been found to be associated with
parenchymal, as well as with vascular amyloid deposits in brains of AD
patients (McGeer and McGeer, 2010).
3.9 Serum Level of IL-1α, IL-10 and IL-17A
3.9.1 Interleukin-1α
Serum level of IL-1α was significantly increased in AD patients (3.79 ±
0.26 pg/ml) as compared with DS patients (2.78 ± 0.39 pg/ml) or controls
(2.78 ± 0.22 pg/ml), while no significant difference was observed between
AD and VD (3.25 ± 0.20 pg/ml) patients or between VD patients, DS
patients and controls (Table 3-24). In addition, distributing the subjects of
Chapter Three: Results and Discussion
۲۲
================================================================
the four investigated groups according to gender, revealed no significant
differences between males and females.
Table 3-24: Serum level of IL-1α in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of IL-1α (pg/ml)
Mean ± SE*
Minimum
Maximum
3.79 ± 0.26A
2.02
9.28
3.25 ± 0.20AB
1.24
4.90
2.78 ± 0.39B
1.21
4.55
B
2.78 ± 0.22
1.02
4.98
*Different letters: Significant difference (P ≤ 0.05) between means.
The present results suggest a role for IL-1α in the pathogenesis of AD.
Interleukin-1α is a pluripotent, pro-inflammatory cytokine that orchestrates
inlfammatory and host defense responses in the peripher�. It also activates T
cells (and, indirectly, B cells), upregulates expression of adhesion molecules,
and induces expression of a number of other pro-inflammatory cytokines and
other inflammation-associated proteins that form an amplifying cascade of
inflammatory response (Arend, 2002). With respect to AD, over-expression
of IL-1. in Alzheimer brain was demonstrated, and such over-expression
was evident both immunohistochemically, as a 6-fold increase in the
numbers of IL-1. -immunoreactive microglia, and biochemically, as elevated
tissue levels of IL-1. . These IL-1. -overexpressing microglia in Alzheimer
brain were frequently associated with A. plaques, and the pattern of
distribution of these microglia across brain regions correlated with the
distribution of A. plaques (Wimo et al., 2006). Such over-expressing
microglia further suggests a role for IL-1. in the initiation and progression of
neuritic and neuronal injury in AD. This association appeared to commence
early in plaque formation, to wax and wane with neuritic pathology within
the plaques (and with the conversion of diffuse Aβ�deposits into compact
Chapter Three: Results and Discussion
۳۳
================================================================
form), and ultimately to disappear in the end-stage “burnt-out” plaques that
are devoid of injured neuritic elements (Butterifel� and
Boyd-Kimball,
2005 ). In AD, even the early, diffuse (non-ifbrillar, and nonneuritic) ‘p�eamyloid’ deposit��were found to contain activated microglia that overexpressing IL-1. . This is in contrast to a lack of microglia in the similar
diffuse A. deposits sometimes found in non-demented elderly individuals;
an observation that suggests that activated microglia may be important in the
initiation of plaque progression and of the neuritic pathology that is central
to the initiation and progression of AD (Parvathy et al., 2009).The
transformation of the presumably benign diffuse deposits of A.�protein into
the diagnostic neuritic plaques of AD was found to be accompanied by
increase in the number, size, and IL-1. immunoreactivity of plaqueassociated microglia, and this was accompanied by progressive condensation
of diffuse A. deposits to form congophilic amyloid (Yao et al., 2011). Due
to such role of IL-1α in AD, the studies hav��
also been extended to shed light
on the association between IL-1α genetic polymorphisms, an��several
authors have reported a significant association between some variants of IL1. gene and AD (Hu et al., 2009; Serretti et al., 2009; Ribizzi et al., 2010; Li
et al., 2013). These studies strongly correlated between the serum level of
IL-1α and its genetic polymorphism and AD, and an implication of suc��
cytokine in the progression of AD can not be ignored.
3.9.2 Interleukin-10
The serum level of IL-10 was approximated in VD and DS patients and
controls (3.39 ± 0.24, 2.77 ± 0.39 and 3.41 ± 0.35 pg/ml, respectively), but
was significantly (P ≤ 0.05) increased in AD patients (5.73 ± 0.55 pg/ml) as
compared to these groups (Table 3-25). In addition, distributing the subjects
of the four investigated groups according to gender, revealed no significant
differences between males and females (data not shown).
Chapter Three: Results and Discussion
٤٤
================================================================
Table 3-25: Serum level of IL-10 in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
30
28
10
20
Serum Level of IL-10 (pg/ml)
Mean ± SE*
Minimum
Maximum
5.73 ± 0.55A
1.04
14.79
3.39 ± 0.24B
1.15
6.07
B
2.77 ± 0.39
0.69
5.12
B
3.41 ± 0.35
0.42
5.43
*Different letters: Significant difference (P ≤ 0.05) between means.
These results suggest that IL-10 (anti-inflammatory and regulatory
cytokine) may play a role in the pathogenesis of AD. In agreement with such
suggestion, Angelopoulos et al. (2008) reported that level of IL-10 is
elevated in the serum of patients with dementia but these levels do not
discriminate between different types of dementia, and one of the mechanisms
attributed to the role of IL-10 in reducing inflammation in AD is suppression
of pro-inflammatory cytokines. Such increase has also been correlated with
Aβ and following immunization with full length Aβ and a corresponding
reduction in plaque load, Tg2576 mice displayed elevated IL-10 plasma
levels. Similarly, mice expressing mutant APP and human presenilin 1
(PS1), immunized with an adenovirus vector encoding repeats of Aβ,
showed increased IL-10 in blood plasma following treatment (Kim et al.,
2007), while a treatment of the mice with granulocyte colony stimulating
factor (GM-CSF) reduced plasma levels of several cytokines, including IL10 (Sanchez-Ramos et al., 2009). Accordingly, monitoring serum level of
IL-10 in AD patients may have therapeutic benefits, but studies of serum
cytokines in AD patients thus far do not have the consistency necessary for a
biomarker, and these preclinical studies suggest that inflammatory markers;
for instance IL-10, may have utility as indicators of therapeutic efficacy
(Sabbagh et al., 2013).
Chapter Three: Results and Discussion
۸٥
================================================================
Interleukin-10 has also been suggested to play an important role in
neuronal homeostasis and cell survival, and mediates its effect on cells by
interacting with specific cell surface receptors (IL-10Rs), present on glial
cell populations in the brain, and it limits inflammation by reducing the
synthesis of pro-inflammatory cytokines such as IL-1α by suppressing
cytokine receptor expression and by inhibiting receptor activation in the
brain (Sabbagh et al., 2013). The regulatory role of IL-10 in AD (and its
correlation with Aβ) has also recently been documented in vitro after
challenging mononuclear cells obtained from AD patients with Aβ. The
results revealed that IL-10 is produced by Aβ-speciifc T helper cells and�
highlight the T-cell-mediated nature of the observed regulatory polarization
of the immune response in Alzheimer patients (Loewenbrueck et al., 2010).
3.9.3 Interleukin-17A
The serum level IL-17A was significantly increased in AD and VD
patients (6.28 ± 0.35 and 5.32 ± 0.42 pg/ml, respectively) as compared with
DS patients (3.75 ± 0.40 pg/ml) or controls (4.05 ± 0.28 pg/ml) (Table 3-26).
In addition, distributing the subjects of the four investigated groups
according to gender, revealed no significant differences between males and
females (data not shown).
Table 3-26: Serum level of IL-17A in Alzheimer's, vascular dementia and
Down's syndrome patients and controls.
Serum Level of IL-17A (pg/ml )
Groups
Alzheimer's disease
Vascular dementia
Down's syndrome
Controls
No.
Mean ± SE*
Minimum
Maximum
30
28
10
20
6.28 ± 0.35A
5.32 ± 0.42A
3.75 ± 0.40B
4.05 ± 0.28B
3.08
2.22
2.08
2.26
11.34
10.69
5.71
6.81
*Different letters: Significant difference (P ≤ 0.05) between means
Chapter Three: Results and Discussion
٦٦
================================================================
Interleukin-17A is pro-inflammatory cytokine secreted by activated Tcells, but recent investigations demonstrated that IL-17A can also be secreted
by innate immune cells such as macrophages, dendritic cells, and NK cells,
and such cytokine emerged as critical players in the pathophysiology of
immune-mediated chronic inflammatory diseases (Heneka and O’Banion,
2007; Korn et al., 2009). Its relation with AD or VD has not well been
investigated, although the present results may suggest a role in both
morbidities. However, Lambracht-Washington et al. (2011) analyzed the
TH17 response in wild-type mice after vaccination with Aβ, and described
for the first time of a TH17 immune response after Aβ peptide immunization.
A direct role for TH17 cells as effector cells causing neuronal dysfunction
and neuroinflammation has recently been described by in vivo imaging
experiments in an EAE mouse model (Siffrin et al. 2010), and it is possible
that Aβ specific TH17 cells might have been involved in the occurrence of
the meningoencephalitis in AD patients; however, further studies are
certainly required to define the role of IL-17A in AD.
3.10 Duration of AD and the Investigated Parameters
To shed light if there was an effect of a disease duration period on the
means of investigated parameters in AD, the patients were distributed into
three groups of duration periods (≤ 5 years, 6-10 years and 11-15 years). A
comparison between the means of each parameter for the three duration
periods was made, and a significant difference was assessed by Duncan test
in which P ≤ 0.05 was considered significant. The results are given in table
(3-27), and they are presented as the following:
1.
Beta-amyloid protein: The mean of Aβ showed a gradual increase as
the disease was progressing through the three durations (30.1 ± 2.9, 53.7
± 5.7 and 68.1 ± 9.8 pg/ml, respectively), but a marked increase was
Chapter Three: Results and Discussion
۷۷
================================================================
observed in the durations 6-10 years and 11-15 years, in which the mean
was significantly increased as compared with the duration ≤ 5 years.
2.
Total cholesterol: The total cholesterol was significantly increased in
the duration ≤ 5 years (240.3 ± 13.7 mg/dL), and then gradually
declined in the durations 6-10 years and 11-15 years (169.9 ± 14.3 and
172.8 ± 22.2 mg/dL, respectively), between which, there was no
significant difference.
3.
Triglycerides: There was no significant difference between the means
of triglycerides in the three investigated duration periods.
4.
Low density lipoproteins cholesterol: The mean of LDL cholesterol
showed a gradual decrease as a duration period was progressing (39.5 ±
1.8, 35.2 ± 0.9 and 30.3 ± 4.1 mg/dL, respectively), and the difference
between the three means was significant.
5.
Low density lipoproteins cholesterol: As in LDL cholesterol, LDL
cholesterol was significantly increased in the first duration period (≤ 5
years: 158.8 ± 14.1 mg/dL), and then it was decreased during the
duration periods 6-10 years and 11-15 years (94.4 ± 15.2 and 108.2 ±
23.4 mg/dL, respectively), but without significant difference between
the latter two means.
6.
Very low density lipoproteins cholesterol: The highest mean of VLDL
cholesterol was observed during the duration period 6-10 years (41.3 ±
3.1 mg/dL), and such difference was significant when the comparison
was made between the mean of the duration period 11-15 years (34.3 ±
2.7 mg/dL).
7.
Total antioxidant capacity: A marked significant decrease in TAC was
observed during the duration period 11-15 years (2.9 ± 0.2 nmol/μL) in
comparison with the means of duration periods 6-10 years and 11-15
Chapter Three: Results and Discussion
۸۸
================================================================
years (7.9 ± 0.5 and 6.2 ± 0.6 nmol/μL, respectively), while there was
no significant difference between the latter two means.
8.
C-reactive protein: The highest mean of CRP was observed during the
duration period 6-10 years (5.7 ± 0.7 mg/dL), and such difference was
significant when the comparison was made between the mean of the
duration period 11-15 years (3.5 ± 0.3 mg/dL).
9.
Alpha 1-antitrypsin: There was no significant difference between the
means of α1-antitrypsin in the three investigated duration periods.
10. Immunoglobulin A: The highest mean of IgA was observed during the
duration period 6-10 years (436.4 ± 36.6 mg/dL), and such difference
was significant when the comparison was made between the means of
the duration periods ≤ 5 years and 11-15 years (348.2 ± 32.2 and 275.8
± 44.4 mg/dL, respectively), while there was no significant difference
between the latter two means.
11. Immunoglobulin G: There was no significant difference between the
means of IgG in the three investigated duration periods.
12. Immunoglobulin M: The serum level of IgM showed a significant
deceased mean in the duration period 11-15 years (120.0 ± 30.1 mg/dL)
as compared with the duration periods ≤ 5 years and 6-10 years (244.7 ±
13.9 and 235.2 ± 25.1 mg/dL, respectively), while there was no
significant difference between the latter two means.
13. Third component of complement: The C3 mean showed a significant
decrease during the duration period ≤ 5 years (139.2 ± 7.2 mg/dl), but it
was increased during the duration periods 6-10 years and 11-15 years
(180.4 ± 12.7 and 208.4 ± 41.4 mg/dL, respectively), while there was no
significant difference between the latter two means.
Chapter Three: Results and Discussion
۹۹
================================================================
14. Fourth component of complement: There was no significant
difference between the means of C4 in the three investigated duration
periods.
15. Interleukin-1α: There was no significant difference between the means
of IL-1α in the three investigated duration periods.
16. Interleukin-10: A significant decrease in the serum level of IL-10 was
observed during the duration period ≤ 5 years (3.7 ± 0.3 pg/ml), and
then, it was increased and approximated during the duration periods 610 years and 11-15 years (5.9 ± 0.8 and 5.9 ± 1.5 pg/ml, respectively).
17. Interleukin-17A: There was no significant difference between the
means of IL-17A in the three investigated duration periods.
Chapter Three: Results and Discussion
۹۰
================================================================
Table 3-27: Means of investigated parameters distributed by duration of
disease in Alzheimer's patients.
Parameter
Beta-amyloid protein (pg/ml)
Total cholesterol (mg/dL)
Triglycerides (mg/dL)
High density lipoproteins
cholesterol (mg/dL)
Low density lipoproteins
cholesterol (mg/dL)
Very low density lipoproteins
cholesterol (mg/dL)
Total antioxidant capacity
(nmol/μL)
C-reactive protein (mg/L)
Alpha 1-antitrypsin (mg/dL)
Immunoglobulin A (mg/dL)
Immunoglobulin G (mg/dL)
Immunoglobulin M (mg/dL)
Third component of
complement (mg/dL)
Fourth component of
complement (mg/dL)
Interleukin-1α (pg/ml)
Interleukin-10 (pg/ml)
Interleukin-17A (pg/ml)
≤ 5 years
30.1 ± 2.9B
240.3±13.7A
194.1±12.3A
39.5 ± 1.8A
Mean ± SE
6-10 years
53.7 ±5.7A
169.9±14.3B
206.8 ±15.5A
35.2 ± 0.9B
11-15 years
68.1 ± 9.8A
172.8 ± 22.2B
171.5 ± 13.7A
30.3 ± 4.1C
158.8±14.1A
94.4 ± 15.2B
108.2 ± 23.4B
38.8 ± 2.5AB
41.3 ± 3.1A
34.3 ± 2.7B
7.9 ±0.5A
6.2 ± 0.6A
2.9 ± 0.2B
4.4 ± 0.4AB
239.5 ±48.5A
348.2± 32.2B
1050.5±119.7A
5.7 ± 0.7A
271.8±29.1A
436.4±36.6A
1206.2±143.7
3.5 ± 0.3B
210.2 ± 71.6A
275.8 ± 44.4B
1352.1 ± 348.3A
244.7± 13.9A
139.2 ±7.2B
235.2± 25.1A
180.4 ± 12.7A
120.0 ± 30.1B
208.4 ± 41.4A
50.9 ±4.9A
48.9 ±3.9A
54.1 ± 6.8A
3.3 ± 0.2A
3.7 ± 0.3B
5.5 ± 0.4A
3.7 ± 0.2A
5.9 ± 0.8A
6.2 ±0.4A
4.2 ± 1.7A
5.9 ±1.5A
6.9 ± 1.3A
A
Different letters: Significant difference (P ≤ 0.05) between means of rows.
The presented results of disease duration in AD patients suggest that
some the investigated parameters might have been impacted by such
duration periods, or the parameter under question might have its effect in a
manner that corresponds to the duration of disease. The first of these
parameters is the Aβ, which was observed with a decreased mean during the
first five years, and then it was gradually increased during the next 10 years.
Chapter Three: Results and Discussion
۹۱
================================================================
Such finding highlights that Aβ paralleled the progression of AD in the
patients, and confirms the crucial role of Aβ in the pathogenesis of AD. In a
recent study, Koyama et al. (2012) conducted a systematic review and metaanalysis of relevant prospective studies to determine whether plasma Aβ
levels may predict development of dementia, AD, and cognitive decline, and
the relationship of plasma Aβ levels to age, dementia status, and cognitive
functioning was also explored. The results revealed that Aβ level was
associated with the pathogenesis of AD in an age-dependent manner.
Furthermore, the authors concluded that plasma Aβ can help in predicting
cognitive function in adults with AD.
Total Cho,HDL-Cho, LDL-Cho level were increased during the first 5
years then gradually decreases during the next 10 and 15 years, there is
obviously an inverse association between βA level and lipid profile level,
while the βA level gradually increased, the lipid profile level gradually
decreased. The explanation for this condition can be made by the concept
that the cholesterol transport through blood occurs by binding to special
protein apoplipoprotein ApoE (HDL, LDL) this protein encodes by ApoE
gene in chromosome 19 by astrocyte in brain (Tang, 2009 ). The ApoE
protein control the concentration of βA depending on the content of fatty
acid, in case of the presence of fatty acid, the ApoE protein decreases the βA
concentration, but in the absent of fatty acid the ApoE protein will increase
the concentration of βA through binding to the βA plaque that make it more
in density (Namba ,etal.,1991). Strittmatter,in 1993 found that ApoE was a
minor protein contaminant that remained tightly bound to βA peptide, also
immunohistochemistry studies demonstrated the presence of ApoE protein in
a high percentage in βA deposits in AD brain tissue (Selkoe,2002) .Patients
after 15 years of disease formation their lipid profile was decreased, this
may be due to malnutrition (anorexia) , but at the contrary the level of βA
was increased.
Chapter Three: Results and Discussion
۲۲
================================================================
Triglyceride is binding with ApoE (VLDL) protein to transport in
blood, it can be transported to all tissues of the body except the brain, brain
not required for triglyceride, it need for cholesterol only ( Murray, et
al.,1996). Hence triglyceride has no affect in βA level.
The TAC showed obvious graduated decrease, this decrease was
associated inversely with gradually increase in βA level. During the first 5
years of disease the level of βA was very low, because the immune system
was not gravely attacked and the ROS and other oxidants which resulted
from the damage of neuron cells can be controlled by antioxidant system.
But during the progression of the disease, that is after 10 and 15 years the
level of βA increases and the immune response for βA become more
effective leading to damage more neuron cells, and the antioxidant system
can not control the accumulation of the oxidants for this reason the TAC
level decreased. The βA accumulation initiate inflammation in the AD brain
resulting in the activation of microglia and the release of neurotoxic
substances, these processes lead to neuronal degeneration, this will
stimulates phagocytosis AD process and independent induces AD (Bing
etal.,2012).
Namba, (1991) mentioned that
several additional proteins are
associated with βA, including, antichymotrypsin, CRP, complement factors,
and immunoglobulins. In our study most very important immunity parameter
(IL-1α, IL-17A,IL-10,IgG,C3 and C4) increased at the third duration of
disease (15 years), this is an important guideline for the attack between βA
and immune system.
Conclusions and Recommendations
۹۲
________________________________________________________________________
Conclusions
Based on the findings of the present study, it is possible to highlight
the following conclusions:
1. Education has certain positive effect on the nerve cells and thus
encouraging the brain function , since most of AD patients were illiterate
(86.7%), while most of VD patients had some sort of education (78.6%).
2. It is possible to use these parameters (Aβ, HDL-ch, LDL-ch, TCA, IL10) to different between the AD and VD patients.
3. There is significant difference in Aβ level between male and female for
AD patients, the level of Aβ higher in female, this mean the disease begin
in female more than male.
4. The parameters (TCA, Aβ , LDL-ch, HDL-ch) useful to use as diagnostic
parameters.
5. the observed increase in Aβ level, with inversely decrease in LDL and
HDL, referred to hypothesis that the most of our AD patients have
ApoE4 gene as essentially cause for AD.
6. the immunity response react with AD in different form depending on
duration of disease .
Conclusions and Recommendations
۹۳
________________________________________________________________________
Recommendations
1. Encourage other studies in this line (Alzheimer's disease) to obtain large
understanding for this disease in our country .
2. Eating healthy food especially food that contain antioxidants like
vegetable, fruit, and coffee.
3. Checking our self every year ( have family history for this disease)
especially after 40s of age for elevated serum level of Beta amyloid
protein.
4. Avoiding some habits that increase the oxidation in the body like
smoking, drinking and eating unhealthy food that contain fat .
5. Perform a genetic study of this disease and observe the correlation
between the genes responsible of AD and beta amyloid level to make
prefect diagnosis for this disease .
6. Establishment of a center for Alzheimer disease.
References
94
___________________________________________________________________________
References:
� Aggarwal, N.T., Bienias, J.L., Bennett, D.A., Wilson, R.S., Morris,
M.C., Schneider, J.A., Shah, R.C. and Evans, D.A. (2006) The relation
of cigarette smokingto incident Alzheimer’s disease in a biracial urban
community population. Neuroepidemiology , 26:140-6.
� Alafuzoff, I., Adolfsson, R., Bucht, G. and Winblad, B. (1983 AugSep). Albumin and immunoglobulin in plasma and cerebrospinal fluid,
and blood-cerebrospinal fluid barrier function in patients with
dementia of Alzheimer type and multi-infarct dementia. Journal of the
neurological sciences, 60(3):465-72.
� Alzheimer’s Association.( 2010). Alzheimer’s disease facts and
figures. Alzheimers Dementia,6: 158-94.
• Angelopoulos, P., Agouridaki, H., Vaiopoulos, H.(2008). Cytokines in
Alzheimer's disease and vascular dementia. The International journal
of neuroscience, 118: 1659-72.
� Anstey, K.J., Lipnicki, D.M., Low, L.F. (2008). Cholesterol as a risk
factor for dementia and cognitive decline: a systematic review of
prospective studies withmeta-analysis. The American journal of
geriatric psychiatry, 16:343-54.
� Anstey, K.J., von Sanden, C., Salim, A. and O’Kearney, R. (2007).
Smoking as a riskfac tor for dementia and cognitive decline: a metaanalysis
of
prospectivestudies.
American
journal
of
epidemiology,166:367-78.
• Anttila, T., Helkala, E.L., Viitanen, M. , Kåreholt, I., Fratiglioni, L.,
Winblad, B., Soininen, H., Tuomilehto, J., Nissinen, A. and
Kivipelto, M. (2004). Alcohol drinkingin middle age and subsequent
risk of mild cognitive impairmentand dementia in old age: a
prospective population based study. British medical journal, 329:539.
� Arend, W.P. (2002). The balance between IL-1 and IL-1Ra in disease.
Cytokine & growth factor reviews, 13:323–40.
� Atwood, C.S., Perry, G., Zeng, H., Kato, Y., Jones, W.D., Ling, K.Q.,
Huang, X., Moir, R.D., Wang, D., Sayre, L.M., Smith, M.A., Chen,
S.G. and Bush, A.I. (2004). Copper mediates dityrosine cross-linking
of Alzheimer's amyloid-beta. Biochemistry, 20;43(2):560-8.
References
95
___________________________________________________________________________
� Babon, J.J., Nicola, N.A. (2012).The biology and mechanism of action
of suppressor of cytokine signaling.Growth Factors, 30(4):207-19.
� Baldeiras, I., Santana, I., Proenca, M.T., Garrucho, M.H., Pascoal, R.,
Rodrigues, A., Duro, D. and Oliveira, C.R. (2008). Peripheral
oxidative damage in mild cognitive impairment and mild Alzheimer's
disease. Journal of Alzheimer’s disease, 15:117–128.
� Bales, K.R., Du, Y., Holtzman, D., Cordell, B. and Paul, S.M. (2000).
Neuroinflammation and Alzheimer's disease: critical roles for
cytokine/Abeta-induced
glial
activation,
NF-kappaB,
and
apolipoprotein E. Neurobiology of aging, 21(3):427-32.
� Ballard,C., Gauthier,S. Corbett, A., Brayne, C., Aarsland,D. and
Jones,E. (2011). Alzheimer’s disease. Lancet, 377: 1019–31.
� Basu, A., Krady, J.K. and Levison, S.W. (2004). Interleukin-1: A
master regulator of neuroinflammation. Journal of Neuroscience
Research, 7: 78(2):151-156.
� Bates, K.A., Verdile, G., Li, Q.X., Ames, D., Hudson, P., Masters,
C.L., Martins, R.N. (2009). Clearance mechanisms of Alzheimer's
amyloid-beta peptide: implications for therapeutic design and
diagnostic tests. Molecular psychiatry, 14(5):469-86.
� Bennett,D.A.,Chneider,J.A.andArvanitakis, Z. (2006).Neuropathology
of older persons without cognitive impairment from two communitybased studies. Neurology. Jun 27;66(12):1837-44.
� Bertram, L., McQueen, M.B., Mullin, K., Blacker, D. and Tanzi, R.E.
(2007). Systematic meta-analyses of Alzheimer disease genetic
association studies: the AlzGene database. Nature genetics,39:17-23
� Bethune, K. (2010). Diagnosis and Treatment of Alzheimer’s Disease:
Current Challenges.Ph.D. Thesis
� Bettens, K., Sleegers, K. and Van Broeckhoven, C. (2013). Genetic
insights in Alzheimer's disease. Lancet Neurol,12(1):92-104.
� Beydoun, M.A., Kaufman, J.S., and Satia, J.A.(2007). Plasma n-3
fatty acids and the risk of cognitive decline in older adults: the
Atherosclerosis Risk in Communities Study. Am J Clin Nutr.
85(4):1103-11.
References
96
___________________________________________________________________________
• Bing-Tian ,B., Huan-Bing, L., Yu-Fang, C., Heng, Z., Ting, L., Ming,
Z. and Zhang , T.(2012). Promotion of b-amyloid production by Creactive protein and its implications in the early pathogenesis of
Alzheimer’s disease Neurochemistry International 60;257–266.
� Blass, D.M. and Rabins, P.V. (2008).In the Clinic: Dementia. Annals
of Internal Medicine. ITC4:1-16.
� Blennow, K., De Leon, M. J.and Zetterberg, H.�
(2006). Alzheimer’s
disease. Lancet, 368: 387–403.
� Bohlson, S. S., Fraser, D. A. and Tenner,A. J. (2007). Complement
proteins C1q and MBL are pattern recognition molecules that signal
immediate and long-term protective immune functions,” Molecular
Immunology, vol. 44, no. 1–3, pp. 33–43,.
� Bonamico, M. (2005). Which is the best screening test for celiac
disease in Down syndrome children?. Journal of pediatric
gastroenterology and Nutrition, 40(2):125-7.
� Bonda, D.J., Wang, X., Perry, G., Nunomura, A., Tabaton, M., Zhu,
X. and Smith, M.A. (2010). Oxidative stress in Alzheimer disease: a
possibility for prevention. Neuropharmacology 59, 290–294.
• Bonotis, K., Krikki, E., Holeva, V., Aggouridaki, C. and Costa, V.
(2008). Systemic immuneaberrations in Alzheimer’s disease patients.
Journal of neuroimmunology,193:183–187.
� Bosco, D., Meda, P., Morel, P., Matthey-Doret, D., Caille, D. (2005).
Expression and secretion of alpha1-proteinase inhibitor are regulated
byproinflammatory cytokines in human pancreatic islet cells.
Diabetologia, 48:1523-33.
� Britschgi, M. and Wyss-Coray, T. (2007). Systemic and acquired
immune responses inAlzheimer’s disease. International review of
neurobiology, 82:205–233.
� Brookmeyer, R., Johnson, E., Ziegler-Graham, K. and Arrighi, H.M.
(2007).�Forecastingthe global burden of Alzheimer’s disease.
Alzheimers Dement, 3:186-191.
• Broughton, S.E., Hercus, T.R., Lopez, A.F. and Parker, M.W. (2012).
Cytokine receptor activation at the cell surface. Current opinion in
structural biology, 22(3):350-9
References
97
___________________________________________________________________________
� Broussard, G.J., Mytar. J., Li, R.C. and Klapstein, G.J. (2012). The
role of inflammatory processes in Alzheimer's disease.
Inflammopharmacology, 20(3):109-26.
� Burgess, B.L., McIsaac, S.A., Naus, K.E., Chan, J.Y., Tansley, G.H.,
Yang, J., Miao, F., Ross, C.J., van Eck, M., Hayden, M.R., van
Nostrand, W., St., George-Hyslop, P., Westaway, D. and Wellington,
C.L. (2006) Elevated plasma triglyceride levels precede amyloid
deposition in Alzheimer's disease mouse models with abundant A beta
in plasma. Neurobiology of disease, 24(1):114-27.
� Butterifeld, D. A. and Boy�-Kimball, D. (2005). The critical role of
methionine 35 in Alzheimer’s amyloid bet�-peptide (1� 42)-induced
oxidative stress and neurotoxicity.
International Journal of
Biochemistry, Biophysics and Molecular Biology,vol. 1703, no.
2:149�156,.
• Butterifeld, D.A., Reed, T.T., Perluigi, M., De Marco, C., Coccia, R.,�
Keller, J.N., Markesbery, W.R. and Sultana, R. (2007). Elevated levels
of 3-nitrotyrosine in brain from subjects with amnestic mild cognitive
impairment: implications for the role of nitration in the progression of
Alzheimer's disease. brain research, 1148: 243�248.
• Buxbaum, J.D., Cullen, E.I. and Friedhoff, L.T. (2002).
Pharmacological concentrations of the HMG-CoA reducatase inhibitor
lovastatin decrease he formation of the Alzheimer beta-amyloid in
vitro and in patients. Front. Biosciences, 7:50–59.
� Cacabelos, R., Franco-Maside, A. and Alvarez, X.A. (1999). Methods
Find ExpClin Interleukin-1 in Alzheimer's disease and multi-infarct
dementia:neuropsychological correlations. Pharmacol, 13(10): 703-8.
� Clavaguera, F., Bolmont, T., Crowther, R.A. ( 2009 ). Transmission
andspreading of tauopathy in transgenic mouse brain. Nature cell
biology, 11: 909–13.
� Cogulu, O., Ozkinay F, Gunduz, C.(2003). Celiac disease in children
with Down syndrome: importance of follow-up and serologic
screening. Pediatrics international, 45(4):395–9.
• Commins, S., Borish, L. and John, W. (2010)Immunologic messenger
molecules: Cytokines, interferons,and chemokines. The Journal of
allergy and clinical immunology , 125:S53-72.
References
98
___________________________________________________________________________
• Corrada, M.M., Brookmeyer, R., Berlau, D., Paganini-Hill, A. and
Kawas, C.H. (2008). Prevalence of dementia after age 90: results from
the 90+ study.Neurology,71:337-343.
�Dacie,J.V. and Lewis, S.(2005). Practical haematology.
6Ed.burgh.churchill.
� David, E., Bernd, K., Jürgen, H. and Kerstin, A. (2008). "Plant metal
chaperones: a novel perspective in dementia therapy". Amyloid-The
Journal of Protein Folding Disorders 16 (2): 81–83.
� Di, Bona D., Plaia, A., Vasto, S., Cavallone, L., Lescai, F., Franceschi,
C., Licastro, F.,Colonna-Romano, G., Lio, D., Candore, G. and
Caruso, C. (2008). Association between theinterleukin-1β
polymorphisms and Alzheimer’s disease: a systematicreview and
meta-analysis. Brain research reviews, 59:155–163.
� Di Bona, D., Rizzo, C., Bonaventura, G., Candore, G. and Caruso, C.
(2012). Association between interleukin-10 polymorphisms and
Alzheimer's disease: a systematic review and meta-analysis. Journal of
Alzheimer’s disease: JAD, 29(4):751-9.
� Di, Bona D., Vasto, S., Capurso, C., Christiansen, L., Deiana, L.,
Franceschi, C., Hurme, M., Mocchegiani, E., Rea, M., Lio, D.,
Candore, G. and Caruso, C. (2009). Effect ofinterleukin-6
polymorphisms on human longevity: a systematic reviewand metaanalysis. Ageing research reviews, 8:36–42.
� Dik, M.G., Jonker, C., Hack, C.E., Smit, J.H., Comijs, H.C. and
Eikelenboom, P. (2005). Serum inflammatory proteinsand cognitive
decline in older persons. Neurology, 64(8):1371–1377.
� Dinarello,C. A. (1997). Induction of interleukin-1 and interleukin- 1
receptor antagonist. Seminars in Oncology, vol. 24, pp. S9- 81–S9-93.
• Dinarello,C. A. (1998). Interleukin-1,interleukin-1 receptors and
interleukin-1 receptor antagonist. International Reviews of
Immunology, vol. 16, pp. 457–499.
Β Ding, J., Eigenbrodt, M.L., Mosley, T.H. Jr, Hutchinson, R.G.,
Folsom, A.R., Harris, T.B. and Nieto, F.J. (2004). Alcohol intake and
cerebral abnormalities onmagnetic resonance imaging in a c
ommunity-based population ofmiddle-aged adults: the Atherosclerosis
Risk in Communities (ARIC)study. Stroke, 35:16-21.
References
99
___________________________________________________________________________
• Dlugai, M., Gerwing, M., Wege, N., Siegrist, J. and Mann,
K.(2012).Elevated levels of high-sensitivity C-reactive protein are
associated with mild cognitive impairment and its subtypes.J
Alzheimers DIS.28(3):503-14.
• Donahue, A.N., Aschner, M., Lash, L.H/, Syversen, T. and Sonntag,
W.E. (2006). Growth hormone administration to aged animals reduces
disulfide glutathione levels in hippocampus. Mechanisms of Ageing
and Development, 127(1):57–63.
� Drummond, G.R., Selemidis, S., Griendling, K.K. and Sobey, C.G.
(2011). Combatingoxidative stress in vascular disease: NADPH
oxidases astherapeutic targets. Nature reviews. Drug discovery,
10(6):453–471.
� Dubois, B., Feldman, H.H., Jacova, C.(2007). Research criteria for the
diagnosis of Alzheimer's disease: revising the NINCDS–ADRDA
criteria. Lancet Neurol.;6:734–46.
� Eisenhardt, S.U., Habersberger, J., Murphy, A., Chen, Y.C., Woollard,
K.J., Bassler, N.,et al . (2009). Dissociation of pentameric to
monomeric C-reactive protein onactivated platelets localizes
inflammation to atherosclerotic plaques. Circulation research,
105:128–137.
� Elovaara, I. (1984). Proteins in serum and cerebrospinal fluid in
demented patients with Down's syndrome. Acta neurologica
Scandinavica, 69(5):302-5.
� Elovaara, I., Palo, J., Erkinjuntti, T. and Sulkava, R. (1987). Serum
and cerebrospinal fluid proteins and the blood-brain barrier in
Alzheimer's disease and multi-infarct dementia. European neurology,
26(4):229-34.
� Engelhart, M.J., Geerlings, M.I., Meijer, J. (2004). Inflammatory
proteins in plasma and the risk ofdementia: The Rotterdam Study.
Archives of neurology, 61(5):668–672.
� Esiri, M.M., Nagy, Z., Smith, M.Z., Barnetson, L. and Smith, A.D.
(1999). Cerebrovasculardisease and threshold for dementia in the early
stages of Alzheimer’s disease. Lancet, 354:919-20.
� Estus, M., Golde, T.E., Ghiso, J., Cheung, T.T., Shaffer, L.M., Mckay,
D.M., Tintner, R., Frangione, B., and Younkin S.G. (1992).
References
100
___________________________________________________________________________
Production of the Alzheimer amyloid b protein by normal proteolytic
processing. Science 258: 126–129.
� Feng, Y. and Wang,X. (2013). Antioxidant Therapies for Alzheimer's
Disease. Oxid Med Cell Longev, 2012: 472932.
� Ferretti, M.T. and Cuello, A.C. ( 2011). Does a pro-inflammatory
process precedeAlzheimer’s disease and mild cognitive impairment?.
Current Alzheimer research, 8:164–174.
� Ferri, C.P., Prince, M., Brayne, C. (2005). Global prevalence of
dementia: aDelphi consensus study. Lancet,366:2112-2117.
� Finch, C.E. and Morgan, T.E. (2007). Systemic inflammation,
infection, ApoE alleles, andAlzheimer disease: a position paper.
Current Alzheimer research, 4:185–189.
� Ford, E.S. and Giles, W.H. (2000). Serum C-reactive protein and selfreported stroke: findings from the ThirdNational Health and Nutrition
Examination Survey. Arterioscler Thromb Vase Biol, 20(4):1052–
1056.
� Forsberg, A., Engler, H., Almkvist, O., Blomquist, G., Hagman,
G.,Wall, A. (2008). PET imaging of amyloid deposition in
patientswith mild cognitive impairment. Neurobiology of aging;
29:1456–1465.
� Fratiglioni, L., Paillard-Borg, S. and Winblad, B. (2004). An active
and socially integratedlifestyle in late life might protect against
Dementia. Lancet neurology, 3:343-53.
� Fratiglioni, L. and Wang, H.X. ( 2000). Smoking and Parkinson’s and
Alzheimer’sdisease: review of the epidemiological studies.
Behavioural brain research,113:117-20.
� Fratiglioni, L., Von Strauss, E. and Qiu CX.(2008). Epidemiology of
the dementias of old age. In Dening T, Jacoby R, Oppenheimer C,
Thomas A, eds. The Oxford Textbook of Old Age Psychiatry. 4th ed.
New York, NY: Oxford University Press: 391-406.
� Freiedewald,W. (1972).cholesterol determination.Clin.Chem.18,499.
� Gallagher, D. AineNí, M., Reisa, A. Sperling, D. and Brian , L.
(2011). Alzheimer’s Disease. O. Hardiman and C.P. Doherty (eds.),
References
101
___________________________________________________________________________
Neurodegenerative Disorders,Springer-Verlag, London Limited pp.4364.
� Gallagher, D., Ni Mhaolain, A., Coen, R. (2010). Detecting prodromal
Alzheimer’s disease in mild cognitive impairment: utility of the
CAMCOG and other neuropsychological predictors. Int J Ger Psych,
25, 1280-87.
� Giovanni, G., Danylaite,V., Leoni, V., Lim, E.T., Ingle, G.T., SastreGarriga, J., Thompson, A.J., Rashid, W., Davies, G., Hillert, J., Miller,
D.H., Bjorkhem, I. and Masterman, T. (2006). Plasma cerebrosterol
and magnetic resonance imaging measures in multiple sclerosis. Clin.
Neurol. Neurosurg. 108,456–460.
� Goedert, M., and Spillantini, G. (2006) A Century of Alzheimer’s
Disease. Science, 314: 777-781.
� Gordon T.(1977). HDL determination.Amer.J.Med.
� Gorelick, P.B., Scuteri, A., Black, S.E., DeCarli, C., Greenberg, S.M.,
Iadecola,C., Launer, L.J., Laurent, S., Lopez, O.L., Nyenhuis, D.,
Petersen, R.C., Schneider, J.A., Tzourio, C., Arnett, D.K., Bennett,
D.A., Chui, H.C., Higashida, R.T., Lindquist, R., Nilsson, P.M.,
Roman, G.C., Sellke, F.W., Seshadri, S. (2011). Vascular
contributions to cognitive impairmentand dementia. Stroke,
42(9):2672–2713.
� Green, R.C., Cupples, L.A and Go, M.. (2002). Risk of dementia
among white and African American relatives of patients with
Alzheimer disease. the journal of the American Medical
Association,287:329-336
� Griffin, W.S. and Barger, S.W. (2010).
Neuroinflammatory
cytokines: the common thread in Alzheimer’s pathogenesis. US
Neurol, 6(2):19-27.
� Griffin, W.S. and Mrak, R.E. (2002). Interleukin-1 in the genesis and
progression of and risk for development of neuronal degeneration in
Alzheimer's disease. Journal of leukocyte biology, 72 (2): 233–8.
� Guilloreau, L., Combalbert, S., Sournia-Saquet, A., Mazarguil, H. and
Faller, P. (2007). Redox chemistry of copper-amyloid-beta: the
generation of hydroxyl radical in the presence of ascorbate is linked to
References
102
___________________________________________________________________________
redox-potentials and aggregation state. Chembiochem, 23;8(11):131725.
� Gupta, A., Watkins, A., Thomas, P. (2004). Coagulation and
inflammatory markers in Alzheimer’s andvascular dementia.
International journal of clinical practice, 59(1):52–57.
� Hardy, J. Has (2006), the amyloid cascade hypothesis for Alzheimer’s
disease been proved? . Current Alzheimer research, 3: 71–73.
� Hayashi, T., Shishido, N., Nakayama, K., Nunomura, A., Smith, M.A.,
Perry, G. and Nakamura, M. (2007). Lipid peroxidation and 4hydroxy-2-nonenal formation by copper ion bound to amyloid-beta
peptide. Free Radical Biology Medicin, 43:1552 –1559.
� Heneka, M.T. and O’Banion, M.K..(2007). Inflammatory processes in
Alzheimer’s disease. J Neuroimmunol. 184:69–91.
� Hill, G., Connelly, J., Hébert, R., Lindsay, J. and Millar, W. (2003).
Neymans bias revisited. J ClinEpidemiol,56:293-6.
� Hoglund, K., Thelen, K.M., Syversen, S., Sjogren, M., von Bergmann,
K., Wallin, A., Vanmechelen, E., Vanderstichele, H., Lutjohann, D.
and Blennow, K. (2005). The effect of simvastatin treatment on the
amyloid precursor protein and brain cholesterol metabolism in patients
with Alzheimer’s disease. Dement. Geriatr. Cogn. Disord, 19:256–
265.
� Honjo, K., van Reekum, R. and Verhoeff, N.P. (2009) Alzheimer's
disease and infection: do infectious agents contribute to progression of
Alzheimer's disease?. Alzheimers Dementia, 5(4):348-60.
� Howlett, D.R., Hussain, I., Powell, D., Tew, D.G., Meek, T.D. and
Chapman, C.(1995). Identification of a novel aspartic protease (Asp 2)
as beta-secretase. Mol Cell Neurosci 14:419–427.
� Hu, J.L., Li, G., Zhou, D.X., Zou, Y.X., Zhu, Z.S., Xu, R.X., Jiang,
X.D. and Zeng, Y.J. (2009). Genetic analysis of interleukin-1A C(889)T polymorphism with Alzheimer disease. Cellular and molecular
neurobiology, 29:81–85.
� Huang, W., Qiu, C., von Strauss, E., Winblad, B. and Fratiglioni L.
(2004). APOE genotype, family history of dementia, and Alzheimer
disease risk: a 6-year follow-up study. Archives of
neurology,61:1930-1934.
References
103
___________________________________________________________________________
� Iadecola, C. (2010). The overlap between neurodegenerative and
vascularfactors in the pathogenesis of dementia. Acta
neuropathologica, 120(3):287–296.
� Iqbal, K., Alonso, A. C. and Chen, S.(2005).Tau pathology in
alzheimer disease and other tauopathies. Biochim Biophys
Acta1739(2-3): 198-210.
� Iwakura, Y., Ishigame, H., Saijo, S. and Nakae, S. (2011). Functional
specialization of interleukin-17 family members. Immunity, 34: 149162.
• James, A.P., Pal, S., Gennat, H.C., Vine, D.F. and Mamo, J.C.L.
(2003). The incorporation and metabolism of amyloidβ into
chylomicron-like lipid emulsions. Journal of Alzheimer’s disease,
5:179–188.
� Janciauskiene, S., Larison, S., Larison, P. (2004). Inhibition of
lipopolysaccharide-mediated human monocyte activation, in vitro, by
alpha1-antitrypsin.Biochemical
and
biophysical
research
Communications, 321: 592–600.
� Joseph, J.A.
Lau,F.C.and Shukitt-Hale, B.(2008).Nutritional
intervention in brain aging: reducing the effects of inflammation and
oxidative stress. Subcell Biochem. 42:299-318.
� Kalaria, R.N., Maestre, G.E., Arizaga, R., et al. (2008). Alzheimer’s
disease and vasculardementia in developing countries: prevalence,
management, andrisk factors. Lancet Neurol,7:812-826.
� Kaur, K., Dhingra, S., Slezak, J., Sharma, A. K., Bajaj, A. and Singal,
P. K. (2009). Biology of TN-Fα and IL-10, and their imbalance in
heart failure. Heart Failure Reviews, 14: 113-123.
� Kay, A.D., May, C., Papadopoulos, N.M., Costello, R., Atack, J.R.,
Luxenberg, J.S., Cutler, N.R. and Rapoport, S.I. (1987). CSF and
serum concentrations of albumin and IgG in Alzheimer's disease.
Neurobiology of aging, 8(1):21-5.
� Kim, S., Song, J., Seungwoo, K., Changsu, H., Moon, H., Youngho
K., Sangmee, A. and Young, K.(2011). Identification of peripheral
inflammatory markers between normal control and Alzheimer’s
disease BMC Neurology 11:51
References
104
___________________________________________________________________________
� Kim, H.D., Tahara, K., Maxwell, J.A., Lalonde, R., Fukuiwa, T.,
Fujihashi, K., Van Kampen, K.R., Kong, F.K., Tang, D.C. and
Fukuchi, K. (2007). Nasal inoculation of an adenovirus vector
encoding 11 tandem repeats of Abeta1-6 upregulates IL-10 expression
and reduces amyloid load in a mo/hu APPswe PS1dE9 mouse model
of Alzheimer's disease. The journal of gene medicine , 9(2):88–98.
� Konstantinos, B., Eleni, K., Vasiliki, H., Christina, A., Vasiliki, C. and
Stavros, B.(2008). Systemic immune aberrations in Alzheimer's
disease patients Journal of Neuroimmunology. 193 ;183–187.
� Korn, T., Bettelli, E., Gao, W., Awasthi ,A., Jager, A., Strom ,T.B.,
Oukka, M. and Kuchroo, V.K.(2009). IL-21 initiates an alternative
pathway to induce proinflammatory T(H)17 cells. Nature.448:484–7.
� Koyama, A., Okereke, O.I, Yang, T., Blacker, D., Selkoe, D.J. and
Grodstein, F. (2012). Plasma amyloid-β as a predictor of dementia and
cognitive decline: a systematic review and meta-analysis. Archives of
neurology, 69(7):824-31.
• Kruman, F. Cholesterol kit.(2002). Neurosci.22(5):1752-1762 (11).
� Kuo, Y..M., Emmerling, M.R., Vigo-Pelfrey, C., Kasunic, T.C.,
Kirkpatrick, J.B., Murdoch, G.H. (1996). Water-soluble Abeta (N-40,
N-42)oligomers in normal and Alzheimer disease brains. The Journal
of biological chemistry, 271: 4077–4081.
� Kusano, C. and Ferrari, B. (2008). Total antioxidant aapacity: a
biomarker in biomedical and nutritional studies. Journal of Cell and
Molecular Biology, 7: 1-15.
� Labrenz,S. R., Calmann, M. A., Heavner, G. A. and Tolman, G.
(2008). The oxidation of methionine-54 of epoetinum alfa does not
affect molecular structure or stability, but does decrease biological
activity. Journal of Pharmaceutical Science and Tec hnol og y, vol. 62,
no. 3: 211–223,.
� Lambracht-Washington, D., Qu, B.X., Fu, M., Anderson, L.D., Jr,
Stüve O., Eagar, T.N. and Rosenberg, R.N. (2011). DNA
immunization against amyloid beta 42 has high potential as safe
therapy for Alzheimer's disease as it diminishes antigen-specific Th1
and Th17 cell proliferation. Cellular and molecular neurobiology,
31(6): 867-74.
105
References
___________________________________________________________________________
� Larbi, A., Pawelec, G., Witkowski, J.M., Schipper, H.M.,
Derhovanessian, E., Goldeck, D. and Fulop, T. (2009). Dramatic
shifts in circulating CD4 but notCD8 T cell subsets in mild
Alzheimer’s disease. Journal of Alzheimer’s disease,17:91–103.
� Ledeboer, A. J., Brev´e, J. P., Wierinckx,A. (2002). Expressionand
regulation of interleukin-10 and interleukin-10 receptorin rat astroglial
and microglial cells. European Journal ofNeuroscience, vol. 16, no.
7,:1175–1185,.
� Lepara, O., Alajbegovic, A., Zaciragic, A., Nakas, E., Valjevac, A.,
Lepara, D., Hadzovic, A., Fajkic, A. and Kulo, A.(2009).Elevated
serum homocysteine level is not associated with serum C-reactive
protein in patients with probable Alzheimer’s diseaseJ Neural Transm
116:1651–1656.
� Li, B.H., Zhang, L.L., Yin, Y.W., Pi, Y., Guo, L., Yang, Q.W., Gao,
C.Y., Fang, C.Q., Wang, J.Z., Xiang, J. and Li, J.C. (2013).
Association between interleukin-1α C(-889)T polymorphism and
Alzheimer's disease: a meta-analysis including 12,817 subjects.
Journal of neural transmission, 120(3):497-506.
� Lim, G.P, Calon, F. and Morihara, T. (2005).diet enriched with the
omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in
an aged Alzheimer mouse model. J Neurosci. Mar 23;25(12):3032-40.
� Lindberg, C., Hjorth, E., Post, C., Winblad, B. and Schultzberg, M.
(2005). Cytokineproduction by a human microglial cell line: effects
ofβ-amyloidandα-melanocyte-stimulating hormone. Neurotoxicity
research, 8:267–276.
� Lindsay, J., Laurin, D., Verreault, R., Hébert, R., Helliwell, B., Hill,
G.B. and McDowell, I. (2002) Risk factors for Alzheimer’s disease:A
prospective analysis from the Canadian study of health andaging.
American journal of epidemiology, 156: 445-453.
� Liu, Y.J., Guo, D.W.,
Peripheral T cells
overexpress CXCR2
which is microglial
31:175–188.
Tian, L., Shang, D.S. and Zhao, W.D. (2010).
derivedfrom Alzheimer’s disease patients
contributing to itstransendothelial migration,
TNF-α-dependent. Neurobiology of aging,
References
106
___________________________________________________________________________
� Llibre, Rodriguez J.J., Ferri, C.P., Acosta, D., et al. (2008). Prevalence
of dementiain Latin America, India, and China: a population-based
cross-sectional survey.Lancet,372:464-474.
� Lobo, A., Launer, L.J., Fratiglioni, L. (2000). Prevalence of dementia
andmajor subtypes in Europe: A collaborative study of populationbasedcohorts. Neurology.;54:S4-S9.
� Locascio, J.J., Fukumoto, H., Yap, L. (2008). Plasma amyloid betaprotein and C-reactive protein inrelation to the rate of progression of
Alzheimer disease. Archives of neurology, 65(6):776–785.
� Loewenbrueck, K.F., Tigno-Aranjuez, J.T., Boehm, B.O., Lehmann,
P.V. and Tary-Lehmann, M. (2010). Th1 responses to beta-amyloid in
young humans convert to regulatory IL-10 responses in Down
syndrome and Alzheimer's disease. Neurobiology of aging,
31(10):1732-42.
� Lott, I., Head, E. and Doran, E.(2006). Beta-amyloid, oxidative stress,
and down syndrome. Curr Alzheimer Res. 3(5):521-528.
� Lovell, M.A. and Markesbery, W.R. (2007). Oxidative DNA damage
in mild cognitive impairment and late-stage Alzheimer’s disease.
Nucleic Acids Research, 35(22):7497–7504.
� Ma, W., Lim, W. and Gee, K. (2001). The p38 mitogen-activated
kinase pathway regulates the human interleukin-10 promoter via the
activation of Sp1 transcription factor in lipopolysaccharide-stimulated
human macrophages. Journal of Biological Chemistry, 276: 1366413674.
� Maes, O.C., Kravitz, S., Mawal, Y., Su, H., Liberman, A., Mehindate,
K., Berlin, D., Sahlas, D.J., Chertkow, H.M., Bergman, H., Melmed,
C. and Schipper, H.M. (2006). Characterization of alpha1-antitrypsin
as a heme oxygenase-1 suppressor in Alzheimer plasma.
Neurobiology of disease, 24(1):89-100.
� Man, S.M., Ma, Y.R., Shang, D.S., Zhao, W.D., Li, B., Guo, D.W.,
Fang, W.G., Zhu, L. and Chen, Y.H. (2007). Peripheral T cells
overexpress MIP-1α to enhance itstransendothelial migration in
Alzheimer’s disease. Neurobiology of aging, 28:485–496.
References
107
___________________________________________________________________________
� Mancinella, A., Mancinella, M., Carpinteri, G. (2009). Is there a
relationship between high C-reactive protein (CRP) levels and
dementia? Arch Gerontol Geriatr .49(Suppl 1):185–194.
� Mancini.(1965) Immunochemistry,2:235.
� Mangialasche, F., Polidori, M.C., Monastero, R., Ercolani, S.,
Camarda, C., Cecchetti, R. and Mecocci, P. (2009). Biomarkers of
oxidative and nitrosative damage in Alzheimer's disease and mild
cognitive impairment. Ageing research reviews, 8:285–305.
� Marklová, E., Albahri, Z. and Vališ, M. (2012) Microheterogeneity of
some serum glycoproteins in neurodegenerative diseases. journal of
neurological Sciences, 15, 314(1-2):20-5.
� Martorana, A., Bulati, M., Silvio, B., Mariavaler O., Calogero, C.,
Giuseppina,C. and Giuseppina C. (2012). Immunosenescence,
inflammation and Alzheimer’s disease. Longevity & Healthspan, 1:818.
� Matthews, F.E., Brayne, C., Lowe, J.( 2009). Epidemiological
pathologyof dementia: attributable-risks at death in the medical
researchcouncil cognitive function and ageing study. PLoS
medicine;6: e1000180.
� Maulik, M., Westaway, D., Jhamandas, J. H. and Kar, S. (2013). Role
of Cholesterol in APP Metabolism and Its Significancein Alzheimer’s
Disease Pathogenesis. Molecular neurobiology, 47:37–63.
� Mayeux, R., Honig, L.S., Tang, M.X., Manly, J., Stern, Y., Schupf,
N.. (2003). Plasma A[beta]40 and A[beta]42 and Alzheimer’s
disease:relation to age, mortality, and risk. Neurology, 61:1185–1190.
� McGeer, P. L. and McGeer,E. G. (2002). The possible role of
complement
activation
in
Alzheimer
disease.
Trends
inMolecularMedicine, vol. 8, no. 11, :519–523,.
� McGeer,E., and McGeer,P. (2010)Neuroinflammation in Alzheimer’s
Diseaseand Mild Cognitive Impairment: A Field in Its Infancy.
Journal of Alzheimer’s Disease , 19 : 355–361.
� Mehta, p.d., Pirttila, T., Mehta, S.P., and Aisen, P.S. (2000). Plasma
and cerebrospinal fluid levels of amyloid peta protein 1-40 and 1-42 in
Alzheimer disease. ARCH NEUROL/VOL 57,JAN.
References
108
___________________________________________________________________________
� Meraz-Ríos, M.A., Lira-De León, K.I., Campos-Peña, V., De AndaHernández, M.A. and Mena-López R. (2010). Tau oligomersand
aggregation in Alzheimer’s disease. Journal of neurochemistry,112:
1353–67.
� Michikawa, M. (2004). Neurodegenerative disorders and cholesterol.
Current Alzheimer research, (4):271-5.
� Michikawa, M. (2006). Role of cholesterol in amyloid cascade:
cholesterol-dependent modulation of tau phosphorylation and
mitochondrial
function. Acta
neurologica
Scandinavica.
Supplementum, 185:21-6.
� Middleton, L.E. and Yaffe, K. (. 2009). Promising strategies for the
prevention of dementia. Archives of neurology, 66(10):1210-5.
� Miscia, S., Ciccocioppo, F., Lanuti, P., Velluto, L. and Bascelli, A.
(2009). Aβ(1–42) stimulate T cells express P-PKC-delta and P-PKCzeta in Alzheimer disease. Neurobiology of aging, 30:394–406.
� Mitra B, Panja, M.( 2005).high sensitive C-reactive protein :anovel
biochemical markers and its role in coronary artery disease,J Assoc
physicians India;53:25-32.
� Mooradian, A.D.(2009). Dyslipidemia in type 2 diabetes mellitus. Nat
Clin Pract Endocrinol Metab. 5(3):150-159.
� Moore, K.W., de Waal, Malefyt R., Coffman, R.L. and O'Garra A.
(2001). Interleukin-10 and the interleukin-10 receptor. Annual Review
of Immunology, 19: 683-765.
� Morris, R. and Mucke, L. (2006). Alzheimer’s disease: a needle
fromthe haystack. Nature, 440: 284–85.
� Moskowitz, M.A., Lo, E.H. and Iadecola, C. (2010). The science of
stroke:mechanisms in search of treatments. Neuron, 67(2):181–198.
� Mrak, R.E. and Griffin, W.S. (2005). Glia and their cytokines in
progression of neurodegeneration. Neurobiology of aging , 26: 349354.
� Namba, Y., Tomonaga, M., Kawasaki, H., Otomo, E. & Ikeda,
K. (1991) Brain Res. 541, 163-166.
References
109
___________________________________________________________________________
� Nakamura, M., Shishido, N., Nunomura, A., Smith, M.A., Perry, G.,
Hayashi, Y., Nakayama, K. and Hayashi, T. (2007). Three histidine
residues of amyloid-beta peptide control the redox activity of copper
and iron. Biochemistry, 46:12737–12743.
� Nerelius, C., Fitzen, M. and Johansson, J. (2010). Amino acid
sequencedeterminants and molecular chaperones in amyloid
fibrilformation. BiochemBiophys Res Commun, 396: 2–6.
• Ness, S., Rafii, M., Aisen, P., Krams, M., Silverman, W. and Manji,
H. (2012). Down's syndrome and Alzheimer's disease: towards
secondary prevention. Nature reviews. Drug discovery, 11(9):655-6.
• Neurochem, Neuroreport,(2002) 13(4):455-459 13.
� Ngandu, T., von Strauss, E., Helkala, E.L., Winblad, B., Nissinen, A.,
Tuomilehto, J., Soininen, H. and Kivipelto, M. (2007). Education and
dementia:what lies behind the association?. Neurology, 69:1442-50.
� Normansell, D.E. (1987). Human immunoglobulin subclasses. Diagn
ClinImmunol, 5:115-28.
� Nunomura, A., Castellani, R.J., Zhu, X., Moreira, P.I., Perry, G. and
Smith, M.A. (2006). Involvement of oxidative stress in Alzheimer
disease. Journal of Neuropathology and Experimental Neurology,
65(7):631–641.
� O’Bryant, S., Waring, S., James, R., Carol, B., Moor, T., Bottiglieri,
P. and Ramon, D. (2010). Decreased C-Reactive Protein Levels in
Alzheimer Disease. Journal of geriatric psychiatry and neurology,
23(1): 49–53.
� Pandey, R.D., Gupta, P.P., Jha, D. and Kumar, S. (2013). Role of
statins in Alzheimer's disease: a retrospective meta-analysis for
commonly investigated clinical parameters in RCTs. The International
journal of neuroscience, 9 (in press).
� Parvathy, S., Rajadas, J., Ryan, H., Vaziri, S., Anderson, L. and
Murphy, GM J.r. (2009). Abeta peptide conformation determines
uptake and interleukin-1alpha expression by primary microglial cells.
Neurobiology of aging, 30(11):1792-804.
� Paul, C.A., Au, R., Fredman, L., Massaro, J.M., Seshadri, S., Decarli,
C. and Wolf, P.A. (2008). Association of alcohol consumption with
References
110
___________________________________________________________________________
brain volume in theFramingham study. Archives of neurology,
65:1363-7.
� Pavlović, M., Arsić, B. and Kazić, B. (2012). Coeliac disease in
children with down syndrome in Serbia. experimental and clinical
research, 13 (1): 9-14.
� Pearson, T.A., Mensah, G.A., Alexander, R.W. (2003). Markers of
inflammation and cardiovasculardisease: application to clinical and
public health practice: a statement for healthcare professionalsfrom the
Centers for Disease Control and Prevention and the American Heart
Association. Circulation, 107(3):499–511.
� Pellicanό, M., Bulati, M., Buffa, S., Barbagallo, M., Di Prima, A.,
Misiano, G., Picone, P., Di Carlo, M., Nuzzo, D., Candore, G., Vasto,
S., Lio, D., Caruso, C. and Colonna-Romano, G. (2010). Systemic
immune responses in Alzheimer’s disease: in vitro mononuclearcell
activation and cytokine production. Journal of Alzheimer’s disease,
21:181–192.
� Pellicanό, M., Larbi, A., Goldeck, D., Colonna-Romano, G., Buffa, S.,
Bulati, M., Rubino, G., Iemolo, F., Candore, G., Caruso, C.,
Derhovanessian, E. and Pawelec, G. (2012). Immune profiling of
Alzheimer patients. Journal of neuroimmunology, 242:52–59.
� Pentland, B., Christie, J.E., Watson, K.C. and Yap, P.L. (1982)
Immunological parameters in Alzheimer's pre-senile dementia. Acta
psychiatrica Scandinavica, 65(5):375-9.
� Perdomo, G. and Dong, H.H. (2009). Apolipoprotein D in lipid
metabolism and its functional implication in atherosclerosis and aging.
Aging, 1:17-27
� Peters, R., Poulter, R, Warner, J., Beckett, N., Burch, L. and Bulpitt,
C. (2008). Smoking,dementia and cognitive decline in the elderly, a
systematic review. BMC geriatrics, 8:36.
� Petersen, R.B., Nunomura, A., Lee, H.G., Casadesus, G., Perry, G.,
Smith, M.A. and Zhu, X. (2007). Signal transduction cascades
associated with oxidative stress in Alzheimer's disease. American
journal of Alzheimer’s disease and other dementias, 11:143–152.
References
111
___________________________________________________________________________
� Phung, T.K., Waltoft, B.L., Laursen, T.M.(2010). Hysterctomy,
oophorectomy and risk of dementia: A nationwide historical cohort
study. Dement Geriatr Cogn Disord, 30:43–50.
� Plassman, B.L., Langa, K.M., Fisher, G.G., et al. (2007). Prevalence
of dementia inthe United States: the aging, demographics, and
memory study.Neuroepidemiology,29:125-132.
� Platzer, C., Docke, W. D.,Volk, H.D. and Prosch, S. (2000).
Catecholamines trigger IL-10 release in acute systemic stress reaction
by direct stimulation of its promoter/enhancer activity in monocytic
cells. Journal of Neuroimmunology, 105: 31-38.
� Pogocki, D. and Schӧneich, C. (2002). Redox properties of Met in
neurotoxic β-amyloid peptide. A molecular modeling study. Chemical
Research in Toxicology, vol. 15, no. 3: 408–418,.
� Poirier, J. (2003). Apolipoprotein E and cholesterol metabolism in the
pathogenesis and treatment of Alzheimer's disease. Trends in
molecular medicine, 9(3):94-101.
� Pomara, N., Willoughby, L.M., Sidtis, J.J. and Mehta, P.D. (2005).
Selectivereductions in plasma Abeta 1–42 in healthy elderly
subjectsduring longitudinal follow-up: a preliminary report. The
American journal of geriatric psychiatry: official journal of the
American Association for Geriatric Psychiatry, 13: 914–917.
� Povova, J., Ambroz, P., Bar, M., Pavukova, V., Sery, O., Tomaskova,
H. and Janout, V. ( 2012). Epidemiological of and risk factors for
Alzheimer's disease: a review.Biomed. Pap Med FacUnivPalacky
Olomouc Czech Repub,156(2):108-14.
� Pratico, D., Clark, C.M., Lee, V.M., Trojanowski, J.Q., Rokach, J. and
Fitz-Gerald, G.A. (2000). Increased 8,12-iso-iPF2α-VI in Alzheimer’s
disease: correlation of a noninvasive index of lipid peroxidation with
disease severity. Annals of Neurology, 48:809–812.
� Puchades, M., Hansson, S.F., Nilsson, C.L., Andreasen, N., Blennow,
K.,Davidsson, P. (2003). Proteomic studies of potential cerebrospinal
fluid protein markers for Alzheimer's disease. Brain Res. Mol. Brain
Res, 118 (1–2), 140–146.
� Puertas, M.C., Martínez-Martos, J.M., Cobo, M.P., Carrera, M.P.,
Mayas, M.D. and Ramírez-Expósito, M.J. (2012) Plasma oxidative
References
112
___________________________________________________________________________
stress parameters in men and women with early stage Alzheimer type
dementia. Experimental gerontology, 47(8):625-30.
� Pulido, R., Jiménez-Escrig, A., Orensanz, L., Saura-Calixto, F. and
Jiménez-Escrig, A. (2005). Study of plasma antioxidant status in
Alzheimer's disease. European journal of neurology, 12(7):531-5.
� Qiu, C., Bäckman, L., Winblad, B., Agüero-Torres, H. and Fratiglioni,
L. (2001). The influenceof education on clinically diagnosed
dementia: incidenceand mortality data from the Kungsholmen Project.
Archives of neurology, 58:2034-9.
� Qiu, C., Kivipelto, M., Aguero-Torres, H. and Winblad, B. and
Fratiglioni, L. (2007). Risk and protective effects of the APOE gene
towards Alzheimer's disease in the Kungsholmen project: variation by
age and sex. J NeurolNeurosurg Psychiatry,75:828-833
� Qiu, C., Kivipelto, M., von Strauss, E.(2009). Epidemiology of
Alzheimer's disease: occurrence, determinants, and strategies toward
intervention. Dialogues ClinNeurosci,11(2):111-28.
� Ravaglia, G., Forti, P., Maioli, F. (2007). Blood inflammatory markers
and risk of dementia: TheConselice Study of Brain Aging.
Neurobiology of aging, 28(12):1810–1820.
� Ray, S., Britschgi, M., Herbert, C., Takeda-Uchimura Y., Boxer,
A.and Blennow K, et al.(2007), Classification and prediction of
clinical Alzheimer’s diagnosis based onplasma signaling proteins.
Nature medicine, 13:1359-1362.
� Reitz, C., Tang, M., Schupf, N., Jennifer, J., Richard, M. and Jose,´
A.(2010). Association of Higher Levels of High-Density Lipoprotein
Cholesterol in Elderly Individuals and Lower Risk of Late-Onset
Alzheimer Disease.Arch Neurol. 67(12):1491-1497.
� Ribizzi, G., Fiordoro, S., Barocci, S., Ferrari, E. and Megna, M.
(2010). Cytokine polymorphisms and Alzheimer disease: possible
associations. Journal of the neurological sciences, 31:321–325.
� Roberson, E.D. and Mucke, L. (2006). 100 years and counting:
Prospects for defeating Alzheimer’s disease. Science, 314:781.
� Roberts, R.O., Geda, Y.E. and Knopman, D.S.(2009). Association of
C-reactive protein with mild cognitive impairment. Alzheimers
Dement 5(5):398–405.
References
113
___________________________________________________________________________
� Rogers, J., Luber-Narod, J., Styren, S.D. and Civin, W.H. (1988).
Expression of immune systemassociatedantigens by cells of the
human central nervous system:relationship to the pathology of
Alzheimer’s disease. Neurobiology of aging, 9:339-349.
� Rubio-Perez, J.M. and Morillas-Ruiz, J.M. (2012). A review:
inflammatory process inAlzheimer’s disease, role of cytokines.
ScientificWorld Journal, 2012:756357.
� Sabbagh, J.J., Kinney, J.W. and Cummings, J.L. (2013). Alzheimer's
disease biomarkers: Correspondence between human studies and
animal models. Neurobiology of disease, 56:116-30.
� Sacco, R.L., Benson, R.T., Kargman, D.E.(2001). High-density
lipoprotein cholesterol and ischemic stroke in the elderly: the Northern
Manhattan Stroke Study. the journal of the American Medical
Association, 285(21):2729-2735.
� Salminen, A., Ojala, J., Kauppinen, A., Kaarniranta, K., and
Suuronen, T.(2009).Inflammation in Alzheimer's disease: amyloidbeta oligomers trigger innate immunity defence via pattern recognition
receptors. Prog Neurobiol .87:181-194.
� Sanchez-Ramos, J., Song, S., Sava, V., Catlow, B., Lin, X., Mori, T.,
Cao, C. and Arendash, G.W. (2009). Granulocyte colony stimulating
factor decreases brain amyloid burden and reverses cognitive
impairment in Alzheimer's mice. Neuroscience, 163 (1):55–72.
� Santarlasci, V., Maggi, L. and Capone, M. (2009). TGF-beta indirectly
favors the development of human Th17 cells by inhibiting Th1 cells.
European Journal of Immunology, 39: 207-215.
� Sardi, F., Fassina, L., Venturini, L., Inguscio, M., Guerriero, F., Rolfo,
E. and Ricevuti, G. (2011). Alzheimer’s disease, autoimmunity and
inflammation. The good, the badand the ugly. Autoimmunity reviews,
11:149–153.
� Schettler, H.( 1975).Nussel,Arb.Med,10,25.
� Schmechel, D.E. (2007). Art, alpha-1-antitrypsin polymorphisms and
intense creative energy:blessing or curse?. Neurotoxicology,
28(5):899–914.
� Schmidt, R., Schmidt, H., Curb, J.D., Masaki, K., White, L.R. and
Launer, L.J. (2002). Early inflammation anddementia: a 25-year
References
114
___________________________________________________________________________
follow-up of the Honolulu-Asia Aging Study. Annals of neurology,
52(2):168–174.
� Schӧneich,C., Pogocki,D., Hug,G.L. and Bobrowski, K. (2003). Free
radical reactions of methionine in peptides: mechanisms relevant to
beta-amyloid oxidation and alzheimer’s disease. Journal of the
American Chemical Society, vol. 125, no. 45: 13700–13713,.
� Selkoe,D.J.(2002). Alzheimer's disease is a synaptic failure. Science,
298:789-791.
� Serretti, A., Olgiati, P., Politis, A., Malitas, P., Albani, D., Dusi, S.,
Polito, L., De Mauro, S., Zisaki, A., Piperi, C., Liappas, I., Stamouli,
E., Mailis, A., Atti, A.R., Morri, M., Ujkaj, M., Batelli, S., Forloni, G.,
Soldatos, C.R., Papadimitriou, G.N., De Ronchi, D. and Kalofoutis, A.
(2009). Lack of association between interleukin-1 alpha rs1800587
polymorphism and Alzheimer’s disease in two Independent European
samples. Journal of Alzheimer’s disease, 16:181–187.
� Seshadri, S., Beiser, A., Kelly-Hayes, M. (2006). The lifetime risk of
stroke: Estimates from the Framingham Study. Stroke, 37:345–350.
� Seubert, P., Vigo-Pelfrey, C., Esch, F. and Lee, M.(1992). Isolation
and quantitation of soluble Alzheimer’s b-peptide from biological
fluids. Nature 359: 325–327, 1992.
• Shen, Y., and Meri, S. (2003). Yin and Yang: complement activation
and regulation in Alzheimer’s disease. Progress in Neurobiology, vol.
70, no. 6: 463–472,.
� Shi, Y. (2012). A human stem cell model of early Alzheimer’s disease
pathology in Down syndrome. Sci. Transl. Med. 4, 124ra29
� Siffrin, V., Radbruch, H., Glumm, R., Niesner, R., Paterka, M., Herz,
J., Leuenberger, T., Lehmann, S.M., Luenstedt, S., Rinnenthal, J.L,,
Laube, G., Luche, H., Lehnardt, S., Fehling, H-J, Griesbeck O. and
Zipp, F. (2010). In vivo imaging of partially reversible Th17 cellinduced neuronal dysfunction in the course of encephalomyelitis.
Immunity, 33(3):424–436.
� Silverman, G.A., Bird, P.I., Carrell, R.W., Church, F.C., Coughlin,
P.B. (2001). The serpins are an expanding superfamily of structurally
similar but functionally diverse proteins. Evolution, mechanism of
References
115
___________________________________________________________________________
inhibition, novel functions, and a revised nomenclature. The Journal of
biological chemistry, 276:33293e6.
� Skoumalová, A. and Hort, J. (2012). Blood markers of oxidative stress
in Alzheimer's disease. Journal of cellular and molecular medicine,
16(10): 2291-300.
� Small, S.A. and Duff K.( 2008). Linking Abeta and tau in late-onset
Alzheimer’s disease: a dual pathway hypothesis. Neuron,60: 534–42.
� Solfrizzi, V., Panza, F., Frisardi, V., Seripa, D., Logroscino, G.,
Imbimbo, B.P. and Pilotto, A. (2011). Diet and Alzheimer's disease
risk factors or prevention: the current evidence. Expert review of
neurotherapeutics, 11(5):677-708.
� Spencer, L.T., Paone, G., Krein, P.M., Rouhani, F.N., Rivera-Nieves,
J. and Brantly, M.L. (2004). Role of human neutrophil peptides in
lung inflammation associated with alpha1-antitrypsin deficiency.
American journal of physiology. Lung cellular and molecular
physiology, 286:L514-L520.
� Srikanth, V., Anderson, J., Donnan, G., Saling, M., Didus, E., Alpitsis,
R, Dewey, H., Macdonell, R. and Thrift, A. (2004). Progressive
dementiaafter first-ever stroke: a community-based follow-up study.
Neurology63, (5) :785–792
� Srikanth, V.K., Quinn, S.J., Donnan, G.A., Saling, M.M. and Thrift,
A.G. (2006). Long-term cognitive transitions, rates of cognitive
change, andpredictors of incident dementia in a population-based firsteverstroke cohort. Stroke, 37(10):2479–2483.
� Stevens, C.D., (Editor). (2010). Clinical Immunology and Serology: a
Laboratory Prospective. F.A. Davis Company, Philadelphia, 54-71.
� Stoltzner, S.E., Grenfell, T.J., Mori, C., Wisniewski, K.E.,
Wisniewski, T.M., Selkoe, D.J. and Lemere, C.A. (2000). Temporal
accrual of complement proteins in amyloid plaques in Down’s
syndrome with Alzheimer’s disease. The American journal of
pathology, 156:489–499.
� Strang, F., Scheichl, A., Chen, Y.C., Wang, X., Htun, N.M., Bassler,
N., Eisenhardt, S., Habersberger, J. and Peter, K. (2012). Amyloid
plaques dissociate pentameric tomonomeric C-Reactive-Protein: a
References
116
___________________________________________________________________________
novel pathomechanism driving corticalinflammation in Alzheimer’s
disease?. Brain pathology, 22:337–346.
� Strle, K. J., Zhou, H., Shen,W. H. (2001). Interleukin-10 in the brain.
Critical Reviews in Immunology, vol. 21, no. 5, : 427–449.
� Strittmatter, W. ANN, M. Saunders, S. Donald, K. Schimefds, A.
Chmechel, D. (1993). Apolipoprotein E: High-avidity binding to ,Bamyloid and increased frequency of type 4 allele in late-onset familial
Alzheimer disease. Proc. Natl. Acad. Sci. USA.Vol. 90, pp. 1977-198.
� Su, S.J., Huang, L.W., Pai, L.S., Liu, H.W. and Chang, K.L. (2008)
Homocysteine at pathophysiologic concentrations activates human
monocyte and induces cytokine expression and inhibits macrophage
migration inhibitory factor expression. Nutrition 21:994–1002.
� Su, J.H., Zhao, M., Anderson, A.J., Sirnivasan, A., Cotman,
C.W.(2001). Activated caspase-3 expression in Alzheimer’s and aged
control brain: correlation with Alzheimer pathology. Brain Res
898:350–357.
� Sultana, R., Piroddi, M., Galli, F., Butterifeld, D.A. (2008). Protein�
levels and activity of some antioxidant enzymes in hippocampus of
subjects with amnestic mild cognitive impairment. Journal of
neurochemistry. Research, 33:2540�2546.
� Sun, Y.X., Minthon, L., Wallmark, A., Warkentin, S., Blennow, K.
and Janciauskiene, S. (2003). Inflammatory markers in matched
plasma and cerebrospinal fluid from patients with Alzheimer's disease.
Dement.Geriatr. Cogn. Disord, 16 (3): 136–144.
� Sundelof, J., Giedraitis, V., Irizarry, M.C., Sundstrom, J., Ingelsson,
E., Ronnemaa, E. (2008). Plasma beta amyloid and the risk of
Alzheimerdisease and dementia in elderly men: a prospective,
populationbasedcohort study. Archives of neurology, 65: 256–263.
� Tan, ZS., Beiser, A.S., Vasan, R.S., Roubenoff, R., Dinarello, C.A.and
Harris, T.B.(2007). Inflammatory markers and the risk of Alzheimer
disease: the Framingham Study. Neurology, 68:1902-8.
• Tang, BL.(2009). Neuronal protein trafifcking associated with�
Alzheimer disease: from APP and BACE1 to glutamate receptors. Cell
AdhMigr 3(1):118�128
References
117
___________________________________________________________________________
� Teunissen, C.E., Scheltens, P. (2007). Use of proteomic approaches to
identify disease biomarkers. Lancet neurology, 6(12):1036-7.
� Thambisetty, M., and Lovestone, S. (2010).Blood-based biomarkers of
Alzheimer’s disease: challenging but feasible Biomark Med. February;
4(1): 65–79.
• Torres, M. and Casadevall, A. (2008). The immunoglobulin constant
region contributes to affinityand specificity. Trends in immunology,
29:91-7.
� Town, T., Kimberly, W.T., Zheng, J.B., Flavell, R.A. and Selkoe, D.J.
(2005). Physiological regulation of the beta-amyloid precursor protein
signalling domain byc-Jun N-terminal kinase JNK3 during neuronal
differentiation. J Neurosci 25:5533–5543.
� Tracy, R.P. (2002). Hemostatic and inflammatory markers as risk
factors for coronary disease in the elderly. The American journal of
geriatric cardiology, 11(2):93–100. [PubMed: 11872967]
� Tyas, S.L., White, L.R., Petrovitch, H., Webster, Ross G., Foley, D.J.,
Heimovitz, H.K. and Launer, L.J. (2003). Mid-life smoking and latelife dementia: the Honolulu-Asia Aging Study. Neurobiology of
aging, 24:589-596.
� Uslu, S., Akarkarasu, Z.E., Ozbabalik, D., Ozkan, S., Colak, O.,
Demirkan, E.S., Ozkiris, A., Demirustu, C. and Alatas, O. (2012).
Levels of amyloid beta-42, interleukin-6 and tumor necrosis factoralpha in Alzheimer's disease and vascular dementia. Neurochemical
research, 37(7):1554-9.
� Van Oijen, M., (2007). Atherosclerosis and risk for dementia. Annals
of neurology , 61:403.
� Van Oijen, M., Witteman, J.C., Hofman, A., Koudstaal, P.J. and
Breteler, M.M. (2005). Fibrinogen is associated withan increased risk
of Alzheimer disease and vascular dementia. Stroke. a journal of
cerebral circulation, 36(12):2637–2641.
� Vance, J.E. (2006). Lipid imbalance in the neurological disorder,
Niemann–Pick C disease. FEBS letters, 580(23):5518–5524.
� Vega, G.L., Weiner, M.F., Lipton, A.M., von Bergmann, K.,
Lutjohann, D., Moore, C. and Svetlik, D. (2003). Reduction in levels
References
118
___________________________________________________________________________
of 24S-hydroxycholesterol by statin treatment in patients with
Alzheimer disease. Arch. Neurological, 60:510–515.
� Vilalta-Franch, J., López-Pousa, S., Calvó-Perxas, L. and Garre-Olmo,
J. (2012). Psychosis of Alzheimer Disease: Prevalence, Incidence,
Persistence, Risk Factors, and Mortality. The American journal of
geriatric psychiatry. official journal of the American, 19. [Epub ahead
of print].
� Wang, H.X., Karp, A., Herlitz, A., Crowe, M., Kåreholt, I., Winblad,
B, and Fratiglioni, L. (2009). Personality and lifestyle in relation to
dementia incidence. Neurology, 72:253-9.
� Wanga, S., Wanga, Y., Su, Y., Zhou, W., Yang, S., Zhang, R., Zhao,
M., Li,Y., Zhang, Z., Zhan, D. and Liu, R.(2012).Rutin inhibits bamyloid aggregation and cytotoxicity, attenuates oxidative stress, and
decreases the production of nitric oxide and proinflamatory cytokines
Neuro Toxicology .33, 482–490.
� Weisman, D., Hakimian, E. and Ho, G.J. (2006). Interleukins,
inflammation, and mechanisms of Alzheimer's disease. Vitamins and
hormones, 74:505-30
� Wilcock, D.M. (2012). Neuroinflammation in the aging Down
syndrome
brain;
lessons
from
Alzheimer's
disease.CurrGerontolGeriatr Res, 2012:170276.
� Wilson, L.M., Pham, C.L., Jenkins, A.J., Wade, J.D., Hill, A.F.,
Perugini, M. A. and Howlett, G.J. (2006). High density lipoproteins
bind Ab and apolipoprotein C-II amyloid fibrils. Journal of lipid
research, 47 (4):755–760.
� Wimo, A., Jonsson, L. and Winblad, B. (2006). "An estimate of the
worldwide prevalence and direct costs of dementia in 2003". Dement
Geriatr Cogn Disord 21 (3): 175–81.
� Wirths, O., Multhaup, G. and Bayer, T.A. (2004). A modified betaamyloidhypothesis: intraneuronal accumulation of the betaamyloidpeptide—the first step of a fatal cascade. Journal of
neurochemistry, 91:513–520.
� Yaffe, K., Kanaya, A., Lindquist, K. (2003). The metabolic syndrome,
inflammation, and risk ofcognitive decline. JAMA: the journal of the
American Medical Association, 292(18):2237–2242.
References
119
___________________________________________________________________________
� Yamada, H. (2010). Current perspectives on the role of IL-17 in
autoimmune disease. Journal of Inflammation Research, 3: 33-44.
� Yao, J.J., He, S.R., Chen, L., Yang, L., Qiao, X.B., Zhang, W., Du, J.
and Liu, D.G. (2011). Expression of cytokine IL-1α and S100β in
different types of plaques in Alzheimer's disease. Zhonghua bing li
xue za zhi Chinese journal of pathology, 40(9):581-4.
� Yao, Z., Painter, S. L. and Fanslow, W. C. (1995). Human IL-17: A
novel cytokine derived from T cells. Journal of Immunology, 155:
5483-5486.
� Yu, H. L., Chertkow, H.M., Bergman, H. and Schipper, H.M. (2003).
Aberrant profiles of native and oxidized glycoproteins in Alzheimer
plasma. Proteomics, 3: 2240–2248.
� Zhou, J., Fonseca, M.I., Pisalyaput, K. and Tenner, A.J. (2008: Sep).
Complement C3 and C4 expression in C1q sufficient and deficient
mouse models of Alzheimer's disease. Journal of neurochemistry,
106(5):2080-92.
� Zhu, X., Lee, H.G., Perry, G. and Smith, M.A. (2007). Alzheimer
disease, the two-hit hypothesis: an update. Acta biochimica et
biophysica Sinica. Acta, 1772:494–502.
� Zhu, X., Raina, A.K., Perry, G. and Smith, M.A. (2004). Alzheimer's
disease: the two-hit hypothesis. Lancet neurology, 3:219–226.
� Zhu, Y., Carvey, P.M. and Ling, Z. (2006). Age-related changes in
glutathione and glutathione-related enzymes in rat brain. Brain
Research, 1090(1):35–44.
� Zigman, W. B. & Lott, I. T. (2007). Alzheimer’s disease in Down
syndrome: neurobiology and risk. Mental Retard. Dev. Disabilities
Res. Rev. 13, 237–246
� Zigman, W., Silverman, W. & Wisniewski, H. M. (1996). Aging and
Alzheimer’s disease in Down syndrome: clinical and pathological
changes. Mental Retard. Dev. Disabilities Res. Rev. 2, 73–79.
Patient Code:
Appendix
Patient's Information Sheet and Laboratory Investigations
Personal Information :
Name:
age:
Address:
Tel. :
Job:
sex:
history:
Smoking:
drinking:
Allergy to fish:
other disease:
Education:
study parameter :
1. IgG:
IgM:
IgA:
2. LDL:
HDL:
VLDL:
3. C3:
C4:
hs CRP:
4. beta amyloid:
5. Total antioxidant capacity:
6. IL-10, IL-17A, IL-1β:
7. α 1att irrypsi::
Notes:
Tri.cho.
Total.ch.
‫ﻼﺨﻼﺻﺔ‬
‫ﺻﻤﻞ ﺑ ﺍﻝﺖﺭﺍﺭﺐ ﺍﻟﺤﺎﻝﺐﻴ ﺑﻠﺪﻑ ﻡ‬
‫‪ .‬ﺗﺸﺨﻴﺺ ﻣﺎﺳﻨ ‪ .‬ﻟﻤﺮﺽ ﺍﻟﺰﺎﻫﺮﻤﻳ ﻭﺗﻤﻣ ﺰﻠﻴ ﻋ ﻟ‬
‫ﺍﻟﺨﺮﻑ ﺍﻝﺎﺷﺊﻨ ﺑﺴﺏﺏ ﺍﻟﺠﻝﻄﺐ ﺍﻟﺪﻣﺎﻏﻴﺔ‪ .‬ﺷﻤﻝ ﺑ ﺍﻝﺖﺭﺍﺭﺐ ﻋﻠﻢ ‪ .۸‬ﻋﻣﻟﺐ ﻞ ﻟ ﺍﻟﻌﺮﺍﻗﻣﻣ ﻟ ﺍ ﻟﻌﺘﺏ ‪.۰‬‬
‫ﻣﺘ‪ .‬ﺰ ﺯﻠ ﺎﻳﻤﺘ ‪ .۹ .‬ﻞ ﺘﻣ ﺰﺟﻠﻄﺐ ﺩ‪.‬ﺎﻏ‪.‬ﺔ‪ .۰ .‬ﻋ‪..‬ﺎ ﺑ ﻞﺑﻼﺯﻣﺐ ﺍﻟﻞﻟﻐﻮﻝﺐﻴ ﻡ‪۰‬ﻼ ﻋﻣﺐﻨ ﺳﻼ‪ .‬ﺘﺐ‬
‫ﻟﻐﺘﺽ ﺍﻟﻤﻘﺍﺘﺔﻧ‪.‬‬
‫ﺟﻤﻌﺖ ﺃﻏﻝﺏ ﻋﻣﻟﺎ ﺑ ﺍﻝﺬ ﺎﻳﻞﺘﻫ ﻡﻋﻣﻟﺎ ﺑ‬
‫‪.‬ﺮﻑ ﺍﻟﺎﺷﺊﻨ ﺑﺴﺒﺐ ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏﺔﻴ ﻣﻦ‬
‫ﻣﺴﺘﺸ ﻔﻢ ﺍﻟﺤﺴﻣ ﻟﺍﻝﺘﻌﻝ ﻣﻤﻴ ﻓﻣﻣﺤﺎﻓﻈﺐ ﻛﺘﺏﻝﺎء‪ .‬ﺃﻣﺎ ﺑﺎﻗ ﻣ ﺍﻟﻌﻣﺎﻨ ﺑ ﺟﻤﻌ ﺑ ﻞ ﻟ ﺩﻭﺘ ﺘﻋﺎﺐﻳ ﺍﻟﻤﺴﻟﻣ ﻟ‬
‫ﻓ‪ .‬ﻛﺮﺑﻝﺎء‪ .‬ﺎﻟﻘﺎﺩﺳﻴﺔ‪ ،‬ﺩﺍﺭ ﺭﻋﺎﺔﻳ ﺍﻟﻤﺴﻨﻴﻦ ﻓﻲ ﻣﺪ‪.‬ﺔﻨ ﺍﻟﺮﺮﺎﺩ ﻭ ﺩﺍﺭ ﺭﻋﺎﺔﻳ ﺍﻟﻤﺴﻨﻴﻦ ﺍﻷﻠﻫﻲ ﺍﻟﺘﺎﺑﻊ‬
‫ﻟﻠﺴﻴﺪ ﺣﺴﻴ� ﺍﻟﺼﺪﺭ ﻓﻲ ﺍﻟﻜﺍﻅﻤﻴﺔ‪ ،‬ﺧﻼﻝ ﺍﻟﻔﺘﺮﺓ ﻣﻦ ﺗﺸﺮﻳﻦﻷﻷﻝﻭ ‪ ۲۰۱۱‬ﺇﻟﻰ ﺣﺰﻳﺮﺍﻥ ‪. ۲۰۱۲‬‬
‫ﺑﻴﺎﻤﻨ ﺟﻤﻌﺖ ﻋﻴﺎﻨﺕ ﺍﻟﻤﻨ ﻐ‪.‬ﻟﺔﻴ ﻓﻘﺪ ﺟﻤﻌﺖ ﻣﻦ ‪.‬ﻌ‪.‬ﺪ ﺍﻟﺮﺟﺎء ﻟﺮﻋﺎﺔﻳ ﺫ ‪ . .‬ﺍﻟﺤﺍﺟﺍﺕ ﺍﻟﺨﺍﺻﺔ ﻓﻲ‬
‫ﻝﺴﻝﻣﻞ‪ .‬ﻟ ﻅﺎﻠﺫﻣﺎ‪.‬‬
‫‪.‬ﻼﻼ ﻼﻼﻼﻼ‪ .‬ﻼ ﻼﻣﻼ ﺑ ﻼ‪.‬ﻼﺫﺐ ﻓﻘﻼ ﺟﻞﻌ ﺑ ﻞﻟﺍﻝﻞﺑﺏ ﺫﻌﻣﻟ ﺍﻝﻌﺸﻡﺍﺋﻣﻣ ﻟ ﺍ‬
‫ﺗﻢ ﺇﺟﺮﺍء ﺑﻌﺾ ﺍﻟﻔﺔ ﺻ‪.‬ﺎﺕ ﺍﻟﺨﺍﺼﺔ ﺑﻤﺼﻞ ﺍﻟﺪﻡ ‪.‬ﻫﻲ ﻛﺎﻵﺗﻲ‪ :‬ﺍﺧﺘﺒﺎﺭ ﺍﻟﻤﺘﻤﻢ ﺍﻟﺜﺎﻟﺚ‬
‫‪.‬ﺍﻟﺮﺍﺑﻊ‪. . . ،α-. . .. ..... . . .. ،‬ﻀﺎﺩ ‪.‬ﻟﻜﻠﺑﻴﻮﻟﻮﻴ ‪ .‬ﺍﻟﻤﻨﺎﻋﻲ ‪.. .. . .. .. ... .. .‬ﺍﻟﺤﺮﻛﺎ‪.‬ﺕ‬
‫ﺍﻟﺨﻠﻮﻳﺔ ‪ ... .. .. .. .. ...... .... .. . .... .. . . .... .1β..‬ﺑﺮﻭﺗ‪.‬ﻦ ‪.. .. .. .. . .. ..‬ﻭ‬
‫‪.‬ﻌﺔ‬
‫ﺍﻟﻜﻠﻴﺔ ﻟﻤﻀﺍﺩﺍﺕ ﺍﻷﻜﻷﺪﺓ ‪.‬‬
‫ﺑﺎﻟ‪.‬ﺴﺒﺔ ﻟﻤﺮﺿﻰ ﺍ ﻟﺰ‪.‬ﺎﻳﻤﺮ‬
‫‪.‬ﺮﺿ‪ ..‬ﺍﻟﺠﻠﻄﺔ ﺍ ﻟﺪ‪.‬ﺎﻏ‪.‬ﺔ ﻛﺎﻥ ‪.‬ﻌﺪﻝ ﺍﻟ ﻌ‪.‬ﺮ ﻟ‪.‬ﻢ ‪ ±‬ﺍﻟﺨﻄﺄ‬
‫ﺍﻟﻘ ﺎﺳ‪.... . ... .. . .. ....... . ... . .. .. .. . ..‬ﺳﻨﺔ ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ(‪ ،‬ﺍ‪.‬ﺍ ﺍﻟ‪.‬ﺪﺓ ﺍ ﻟﺰ‪.‬ﻨﺔ‪ .‬ﻟ ﻠ‪.‬ﺮﺽ ﻓ ‪,‬‬
‫‪.(�.‬‬
‫‪.‬ﺮﺿ‪ .‬ﺍﻟﺨﺮﻑ ﻛﺎﻧﺖ )‪ ۱٥-٦‬ﺳﺔﻨ( ﺑﻴﻨﻤﺎ ﻓﻲﻣﻀ ﻰ ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏﻴﺔ ﻛﺎﻧﺖ )‪ ≤ ٥‬ﺳ‬
‫ﺑﻴﻦ ﻣﺮﺿﻰ ﺍﻟﺰﻫﺎﻳﻤﺮ ﺍﻋﻠﻲ ﻣﻌﺪﻝ ﻟﻤﺴﺘﻮﻯ ﺑﺮﻭ‪.‬ﻴ ‪.‬‬
‫‪ . . ... .. . .. ..‬ﻓﻲ ﺍﻟ‪.‬ﺼﻞ‬
‫‪ .. .. . . .. . .. . . . . .. ..‬ﺑ‪.‬ﺎﻤﻨ ﻛﺎ ‪ .‬ﻣﻌﺪﻝ ﻫﺬﺍ ﺍﻟﺒﺮﻭﺗﻴ� ﻓﻲ ﻣﺼﻞ ﻣﺮﺿﻰ ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏ‪.‬ﺔ‬
‫) ‪. .. . ..... . .. . .. . .. .‬‬
‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﻔﺤﺺ ﺩﻫ ‪.‬ﻥ ﺍﻟﺪﻡ ﻓﻘﺪ ﺍﻅﻬﺮ ﻣﺮﺿﻰ ﺍﻟﺠﻠﻄﺔ ﺍ ﻟﺪ‪.‬ﺎﻏﺔ‪ .‬ﻋﻠﻰ ﺃ ﻋﻠ‪. .‬ﻌﺪﻝ‬
‫ﻟﻜ‪.‬ﻟﺴﺘ‪ .‬ﺮ‪.‬ﻝ ﺍﻟﺪﻡ ‪ .. . . ... . . .. . .. . ..‬ﺣﻴﺚ ﺷﻜﻞ ﻓﺮﻕ ﻣﻌﻨﻮ ‪ .‬ﻣﻊ ﻣﺮﺿﻰ ﺍﻟﺰﺎﻳﻤﺮﻫ ‪.. . . .‬‬
‫‪، .. . .. . . . . .. . .‬ﺃﻣﺎ ﻓﺤﺺ ﺍﻟﺪﻫﻮﻥ ﺍﻟﺜﻼﺛﻴﺔ ﻓﻠﻢ ﻳﺸﻜﻞ ﻓﺮﻕ ﻣﻌﻨﻮﻱ ﺑﻴﻦ ﻣﺮﺿﻰ ﺍﻟﺰﺎﻫﻳﻤﺮ‬
‫ﻣﺮﺿﻭﻰ ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏﻴﺔ ‪ (. . . ... . .. . . .. . . .. . .. . .. . ..‬ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ ‪،‬ﺃﻣﺎ ﻼ‪.‬ﺋﺞ ﻓﺤﺺ‬
‫ﺍﻟﺪﻫﻮﻥ ﻋﺎﻟﺔﻴ ﺍﻟﻜﺜﺎﻓﺔ ﻓﻘﺪ ﺃﺷﺎﺭﺕ ﺇﻟﻰ ﺯﺎﻳﺩﺓ ﻣﻌﻨﻮﺔﻳ ﻋﻨﺪ ﻣﻘﺎﺭﻧﺔ ﻛﻞ ﻣﻦ ﻣﺮﺿﻰ ﺍﻟﺰﺎﻫﺮﻤﻳ ﻣﺮﻭﺿﻰ‬
‫ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏﺔﻴ ‪ . . .. ... . .. ... . ... .. . .. ... . .. . .. ... . .. .. . .. . .‬ﻋﻠﻰ ﺍﻟﺍﻮﺘﻟﻲ( ﺑﻌﻴﻨﺎﺕ‬
‫ﺍﻟﺴﻴﻄﺮﺓ ‪... . ..... . .. .. . .. . .‬‬
‫ﺃﻣﺎ ﻓﺤﺺ ﺍﻟﺪﻫﻮﻥ ﻣﻨﺨﻔﻀﺔ ﺍﻟﻜﺜﺎﻓﺔ ﻓﻘﺪ ﻟﺣﻆﻮ ﺃﻋﻠﻰ ﻣﻌﺪﻝ ﻟﻬﺎ ﻓﻲ ﻣﺮﺿﻰ ﺍﻟﺠﻠﻄﺔ‬
‫ﺍﻟﺪﻣﺎﻏﺔﻴ ‪.. . . .. ... . . ... . . .. . ...‬‬
‫ﺍﻟﺰﺎﻫ ‪..‬ﺮ ) ‪. . .. .‬‬
‫‪.‬ﻗﺪ ﺷﻜﻠﺖ ﻓﺮﻗﺎ ﻣﻌﻨﻮﻱ ﻋﻨﺪ ﻣﻘﺎﺭﻧﺘﻬﺎ ﺑﻌﻴﺎﻨﺕ ﻣﺮﺿﻰ‬
‫‪... . .. .. ....‬‬
‫ﻝﻜﻝﻣﺐ ﻟﻞﺰﺎﺩﺍ ‪.‬‬
‫‪..‬ﺴ ‪ .‬ﻟﻔﺤ ‪ .‬ﺍﻟﺴﻌﺐ ﺍ‬
‫‪..‬ﺩﺐ ﻓﻘﺩﻝﻡﺣﺿ ﺍﻗﻝ ﻞﻌﺩﻝ ﻓ‪.‬‬
‫ﻞﺘﺰﻢ ﺍﻝﺬﻠﺎﻣ ‪ .‬ﺘ‬
‫)‪ .5.29 ± 0.46 nmol/μL‬ﻣﻘﺎﺭﻧﺔ ﻣﻊ ﻣﺮﺿﻰ ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏﺔﻴ ‪.(8.85 ± 0.40 nmol/μL),‬‬
‫ﺃﻣﺎ ﻓﺤﺺ ‪ . .. .... ... .. . .. .. .. .‬ﻓﻘﺪ ﻛﺎﻥ ﺃ‪ �.‬ﻣﻌﺪﻝ ﻟﻪ ﻓﻲ ﻣﺮﺿﻰ ﺍﻟﺰﺎﻫﻳﻤﺮ‬
‫ﺍﻭﻟﺠﻠﻄﺔ ‪.‬ﻟﺪﻣﺎﻏﺔﻴ ‪ .. .. . .. .. .. . .. . . .. .. . ... . .. . .. . .. .‬ﻋﻠﻰ ﺍﻟﺘﻮﻟﺍﻲ(‪ .‬ﻓﺤﺺ ‪α 1.‬‬
‫‪. .. ..... . . ..‬ﺎﻥ ﺃﻋﻠﻰ ‪.‬ﻌﺪﻝﻟ ﻪ ﻣﺮﺿ ﻲﻓﻰ ﺍﻟﺰﺎﻫﻳ ﺮ‪. .. . . .. .. . .. . .. . ... .‬‬
‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﻔﺤﺺ ﺍﻟﻜﻠﻮ‪.‬ﻴﻮﻟﻴ ‪ .‬ﺍﻟ‪.‬ﺎﻨ ﻋ‪ .. . .‬ﻻ‪ .‬ﻻ ﻣﻌﺪﻻﺗﻪ ‪.‬ﺘﻘﺎﺭﺑﺔ ﻓﻲ ﻣﺮﺿﻰ ﺍﻻﺰﻻ‪. ..‬ﻻ‬
‫ﻣﺮﺿﻢﻭﻻﻟﺠﻝﻄﺐ ﺍﻻ‪.‬ﻻﻏﻻ‪.... . . ... . . .، .. . . ... . . .. . .. ) .‬ﻋﻠﻰ ﺍﻟﺘﻮﺍﻟﻲ‪ ،‬ﺃﻣﺎ ﺍﻟﻜﻠﻮﻻ‪. .‬ﻻ‪. .‬‬
‫ﺍﻟﻤﺎﻨﻋﻲ ‪. . .. .‬ﻓﻠﻢ ﻈﻳﺮﻬ ﻓﺮﻕ ﻣﻌ‪.‬ﻮﻱ ﺑﻴﻦ ﻣﺮﺿﻰ ﺍﻟﺰﺎﻳﻤﺮﻫ ﻻ‪.‬ﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏ‪.‬ﺔ ﺑﻴﻨﺎﻤ ﻛﺎﻧ ﻻ‬
‫�ﺎﺕ‪.‬‬
‫‪T‬ﻻﺖﻝﺍ ﻻ ﺍ ﻻﻮﺑ‪.‬ﻮﻻ‪ ..‬ﺍﻟﻤﻻ‪.‬ﻋﻲ ‪ .. .‬ﻣﺘﻘﺎﺭﺑﺔ ﻟﺠﻤﻊﻴ ﺍﻟﻌ‪.‬‬
‫ﺑﺎﻟﻨﺴﺒﺔ ﻟﻔﺤﺺ ﺍﻟﻤﺘﻤﻢ ﺍﻟﻨﻤﻂ ﺍﻟﺜﺎﻟﺚ ﻭﺍﻟﺮﺍﺑﻊ ﻛﺎﻧﺖ ﺃﻋﻠﻰ ﺴﻧﺒﺔ ﻟﻪ ﻓﻲ ﻣﺮﺿﻰ ﺍﻟﺰﺎﻳﻤﺮﻫ ‪. . . .‬‬
‫‪. (. . .. ... . .. .. . .. . .... ... .‬ﺑﺎﻟﻨﺴﺒﺔ ﻟﻔﺤﺺ ‪.‬ﻟﺤﺮﻛﺎﻴﺕ ﺍﻟﺨﻠﻮﺔﻳ ‪ .. .1α,IL.. . .‬ﻓﻠﻢ ﻜ‪. .‬‬
‫ﺎﻙﻨﻫ ﻓﺮﻕ ﻣﻌﻨﻮﻱ ﺑﻴ� ﻣﺮﺿﻰ ﺍﻟﺰﻫﺎﻳﻤﺮ ﻣﺮﺿﻭﻰ ﺍﻟﺠﻠﻄﺔ ﺍﻟﺪﻣﺎﻏﺔﻴ‪ ،‬ﺑﻴﻨﻤ‪ .‬ﻛﺎﻧﺖ ﺎﻙﻨﻫ ﺯﺎﻳﺩﺓ‬
‫ﻣﻠﺤﻅﺔﻮ ﻓﻲ ﻣﻌﺪﻻﺕ ‪ .. .. . .‬ﻣﺮﺰﻢ ﻲﻓ ﻻ‪ñ‬ﺎﻳﻤﺮ ‪. .. ... .. ... . . .. . .. ..‬‬
‫�ﺍﻻﻛﺴﺪﺓ ﻟﻠﺘﻤﻴﺰ‬
‫ﺗﻮﺻﻝ ﺑﺍﻝﺩﺭﺍﺭﺐ ﺇﻟﻢ ﺃﻫﻞﺐﻴ ﻛﻝ ﻞ ﻟ ﺑﺘﻡﺑﻣ ﻟﺍﻝﺏﻣﻻ ﺍﻣﺍﻳﻠﻮﺩ ﻭﺍﻟﺴﻌﺔ ﺍﻟﻜﻠﺔﻴ ﻟﻤﻈﺎﺩﺍﺕ‬
‫ﻣﻀﻢ ﻦﻴﺑ ﺍ ﻟﺨﺘ ﻑ ﻭﻣﺘ ﺰ ﻢﻻﻟﺠﻠﻄﺐﻻﻟﺪﻣﺎﻏﺐﻴ ‪.‬‬
‫‪.‬‬
‫جمهورية العراق‬
‫وزارة التعليم العالي والبحث العلمي‬
‫جامعة بغداد ــ كلية العلوم‬
‫قسم التقنيات اإلحيائية‬
‫النسق املناعي ملرض الزهامير يف عينة من‬
‫املرضى العراقيني‬
‫ﺃﻁﺮﻭﺣﺔ‬
‫ﻣﻘﺪﻣﺔ ﺇﻟﻰ ‪.. ....‬ﻦ‪ .‬ﺎ‪...‬ﻢ‪/ .‬ﺟ ﺎ‪ ...‬ﺑﻐ ‪ ...‬ﻫﻭﻤ ﺟﺰء ﻣﻦ ﻣ ‪..‬ﺒﺎﺕ‬
‫ﻹﻣﻦﻹ‪ /.‬ﺍﻹﻤﻢﺎﻹﻹ‬
‫ﻧﻴﻞ ﺩﺭ ‪.... ..‬ﺕﻢ ﺘﺍﻣ ‪ .....‬ﻓ ﻤ ‪..‬ﺘﻘﻢﻦ‪.‬ﺕﻹﻹﺣ‬
‫ﺗﻘﺪ ﻹ ﻹﻬﺍ ‪:‬‬
‫ﺁﻻء ﻻﺒﺪ ﺍﻟﺤﺴﻦ ﻻﻤﻻﻼﻥ ﺍﻟﻜﻨﺰ ﻻﻱ‬
‫ﻻﻻﻻﻻﻭﻻ ‪/‬ﻻﻻﻻﻻﻻ ﻻﻣﻻﻻﻻ)‪(۲٦٦٦‬‬
‫ﻻﻻﻢﻻﻥ ﻢﻻ ﻻﻻﻢﻡ ﺤﻴﻻﻻ ‪/‬ﻻﻻﻣﻻ‬
‫ﺍﻻﻻﻻﻻﻻ ‪ /‬ﻻﻻﻻﻻﻻﻻﻻﻻﺍﻻ )‪(۲۰۹۹‬‬
‫ﻣﺎﺟﺴﺘﻴﺮ ﻻﻘﻨﻴﺎﺕﻻﺣﻴﺎﻱﻴﺔ‪ /‬ﻻ‪.‬ﻻ‬
‫ﻻﻻ ﻻﻻ ﻻ‬
‫ﺩ‪..‬ﺃ‪.‬ﺃﻟ ‪.‬ﻦﻛﺮﻥﻜﻢﺭ ﺁ ﻏﻢﺏ‬
‫�ﺍﻟﺤﺠﺔ ‪.‬ـ ‪۱٤۳٤‬‬
‫ﺫﻣ‬
‫ﺃ‪ .‬ﺩ‪ .‬ﻋﻠ ﻤﺣﺴﻦﻦ ﺃﺩﺣﻦﺔ‬
‫ﺗﺸﺮﻥﻦ ﺍﻟﺜﺍﻧﻤ ‪۲۰۱۳.‬‬