et al(2003)

Urinary Glycosaminoglycans (GAG) In Patients
With Systemic Lupus Erythematosus (SLE):
Marker Of Renal Affection And Relation To
Disease Activity
Thesis
Submitted in Partial Fulfillment for
Requirement of the degree of
M.Sc. of Rheumatology & Rehabilitation
Prof. Dr.
Heba Abd El- Wahab Seliem
Assistant professor of Rheumatology &
Rehabilitation
Faculty of Medicine
Zagazig University
Prof. Dr.
Omayma Zakaria Shehata
Assistant professor of Rheumatology &
Rehabilitation
Faculty of Medicine
Zagazig University
By
Samah Fathy Abas Hamad
M.B.B.ch
Faculty of Medicine-Zagazig University
Under supervision of
Prof. Dr.
Yousry Abu El Magd
Professor of Biochemistry
Faculty of Medicine
Zagazig University
Dr.
Ahmed Abd El-Monem Emera
Lecturer of Rheumatology &
Rehabilitation
Faculty of Medicine
Zagazig University
Faculty of Medicine
Zagazig University
2004

ميحرلا نمحرلا ﷲ مسب
ِﻪ ْ ﻴَﻠَﻋ ِﻪﱠﻠﻟﺎِﺑ ﱠﻻِإ ﻲِﻘﻴِﻓ ْ ﻮ َ ﺗ ﺎ َ ﻣ َ و
ﺐﻴِﻧُأ ِﻪ ﻴَﻟِإ
ُ ْ و َ ﺖْﻠﱠﻛ ُ ﻮَ َﺗ ميظعلا ﷲ قدص
(
٨٨ةيلآا
نم : دوھ)

Dedication
To my parents
To my sisters and brother
To my husband and son

Acknowledgement
First of all, 1 am indebted in all work to Gracious Allah.
All thanks and deep gratitude are to professor Dr. Heba Abd EL-
Wahab Seliem. Ass. Prof. of Rheumatology, Rehabilitation Faculty of
Medicine. Zagazig University. I will never forget her kind supervision
and endless help through the whole study and beneficial instructions on
the final touches of this thesis.
Many thanks and deep gratitude to professor Dr. Yousry Abu EL-
Magd. Prof. of Biochemistry Faculty of Medicine. Zagazig University for
his great help, precious advice and supervision.
Great thanks are given to professor Dr. Omayma Zakaria Shehata.
Ass. Prof of Rheumatology & Rehabilitation Faculty of Medicine.
Zagazig University for her valuble help and supervision throughout this
work.
My deepest appreciation is to Dr. Ahmed Abd El- Monem Emera.
Lecturer of Rheumatology & Rehabilitation Faculty of Medicine. Zagazig
University for his valuable help and guidance through out this work.
It is my pleasure to thank all the staff of my department, for their
general support and advice during the work.
Finally, I will never forget to thank all my patients to whom this
study was carried out. Without their cooperation this thesis was never
going to appear.

List of abbreviations
AECA Antiendothelial cell antibodies
ANA Antinuclear antibodies
apl Antiphospholipid
Apo-A-1 Apolipoprotein A-1
APS Antiphospholipid Syndrome
ARA American Rheumatism Association
BILAG British Isles Lupus Assessment group.
C3
Complement 3
C3d Complement 3 d
C4
Complement 4
CD Cystidine deaminase
CL Cardiolipin
CNS Central nervous system
CRP C-reactive protein
CS Chondriotin sulfate
CS PG Chondriotin sulfate proteoglycans
DNA Deoxy Nucleic acid
DS Dermatan sulfate
Ds. DNA Double stranded deoxy nucleic acid
EC Endothelial cell
ESR Erythrocytic sedimentation rate
FGE Functional GAG excretion
GAGs Glycosaminoglycans
- I-

GaINAC N-acetyl galactosamine
Gal Galactose
GBM Glomerular basement membrane
GIcUA Glucuronic acid
HA Hyaluronic acid
HDL High density lipoprotein
HLA DR2
HLA DR3
Human leucocyte antigen DR2
Human leucocyte antigen DR3
HMG High morbidity group
HS Heparan sulfate
HS PG Heparan sulfate proteoglycans
IgG Immunoglobulin G
IGM Immunoglobulin M
IL2
Interlukin 2
IL-6 Interleukin 6
KS Keratan sulfate
LAC Lupus anticoagulant
LL Lower Limb
LN Lupus Nephritis
LTD4
LTE4
Leukotrein D4
Leukotrein E4
MHC Major histocompitability complex
OA Osteoarthritis
PAPs 3- phosphoadenosine 5-phosphosulfate
- II-

RA Rheumatoid arthritis
RF Rheumatoid factor
RNP Ribonucleoprotein
SLAM Systemic lupus activity measures
SLE Systemic Lupus Erythematosus
Sm RNP Small nuclear ribonucleo protein
SS DNA Single stranded DNA
STNFR Soluble tumour necrosis factor receptor
UCH University college hospital
US United states
UV Ultraviolet
XYL Xylose
Z DNA Left handed DNA
- III-

List of tables
Tables of Review and (subject and Methods)
Table I Frequency of various clinical manifestations at
presentation or at any time during the course of
SLE
-IV
-
page
Table II The different conditions with positive ANA 13
Table III The ANA patterns of different diseases 17
Table IV Renal immunopathalogy 24
Table V Correlation between clinical and laboratory
findings and pathologic classifications in lupus
nephritis patients.
Table VI Proposed immunopathogenic mechanisms in
lupus nephritis
Table VII Additional evidence for the in vivo Relevance of
Nucleosome- mediated binding of auto antibodies
to the glomerular basement membrane in SLE
nephritis.
Table VIII Summary of proteogly can properties. 54
Table IX The UCH/ Middle sex score. 67
4
28
30
33

Tables of the Results:
-V
-
Page
Table 1 Demographic data of SLE patients and control group 79
Table 2 Subgroups of SLE patients as regard the disease activity
index.
Table 3 Subgroups of SLE patients as regard renal involvements 80
Table 4 Clinical characteristics of disease activity in SLE patients. 81
Table 5 Clinical parameters of disease activity in SLE subgroups. 82
Table 6 Laboratory findings among SLE patients 83
Table 7 Laboratory findings of SLE subgroups 84
Table 8 Urinary GAG , HS and CS in SLE patients 85
Table 9 CS and HS levels in SLE subgroups and control group. 85
Table 10 Comparison between urinary GAG level and clinical
parameters of disease activity in SLE patients.
Table 11 Correlation between urinary GAG and other laboratory
parameters of disease activity.
Table 12 Comparison between SLE patients group and control
group as regard total urinary GAG level
Table 13 Comparison between SLE patients and control group as
regard Hs and Cs level
Table 14 Comparison between SLE patients and control group as
regard Cs/Hs ratio.
Table 15 Ratio between Cs/Hs in Active and inactive SLE patients. 89
Table 16 Comparison between SLE subgroups (according to renal
affection) and control group as regard urinary GAG
Table 17 Comparison between SLE subgroups and control as
regard Cs and Hs levels in relation to renal involvement.
Table 18 Comparison between SLE subgroups (regarding renal
affection) & control group as regard Cs/Hs ratio.
80
86
87
87
88
89
90
91
92

List of Figures
-VI
-
Page
Figure I Structure of proteoglycan 41
Figure II Synthesis of chondriotin sulfate proteoglycan 43
Figure III Repeat unit of hyaluronic acid 44
Figure IV The repeating disaceharide units of (a) chondriotin
4-sulfate and (b) chondriotin 6- sulfate
Figure V Heparin and Heparan sulfate (Hs) 48
Figure VI Repeat unit of dermatan sulfate 51
Figure VII Repeat unit of keratan sulfate. 52
Figure VIII Clinical characteristics of disease activity in SLE
patients
Figure IX Correlation between urinary GAG and laboratory
parameters of the disease activity.
46
80
87

CONTENTS
Page
Introduction ......................................................................... 1
Aim of the work .................................................................. 3
Review of literature .......................................................... 4
Systemic lupus erythematosus .......................................... 4
Epidemiology ........................................................................ 6
Etiology and pathogenesis ..................................................... 7
Immunopathogenesis .......................................................... 10
Immunopathology ................................................................ 21
Lupus nephritis ..................................................................... 25
Assessment of SLE activity ................................................ 34
Activity indices .................................................................. 34
Laboratory markers of disease activity in SLE ................. 35
Glycosaminoglycans ........................................................... 40
Subjects and methods .................................................... 60
Results .................................................................................. 75
Discussion ............................................................................ 93
Summary and conclusion .......................................... 100
References ......................................................................... 106
Appendix ........................................................................... 129
Recommendation
Arabic summary

Introduction & Aim of the Work
Introduction
Systemic lupus erythematosus is an autoimmune disease
characterized by loss of immunologic self-tolerance and the
subsequent development of auto antibodies. (Gehi, et al 2003).
This autoimmune process plays a crucial role in the
pathogenesis of SLE, (Kozak, et al., 2000).
Autoantigens in systemic lupus erythematosus are highly
diverse in terms of structure and location in control cells. (White
and Rosen, 2003). Lupus nephritis (LN) remains a leading
cause of morbidity and mortality in SLE. Progression to LN in
SLE is dependent on the host breaking immune tolerance and
forming autoantibodies that deposit in the kidney (Oates, and
Gitkeson, 2002).
Glycosaminoglycans (GAGs) are highly negatively
charged molecules, with extended conformation that imparts
high viscosity to the solution. GAGs are located primarly on the
surface of cells or in the extracellular matrix (ECM). Along with
the high viscosity of GAGS comes low compressibility, which
makes these molecules ideal for a lubricating fluid in the joints.
١

Introduction & Aim of the Work
At the same time their rigidity provides structural integrity
to cells and provides passage ways between cells. Allowing for
cell migration (Michael and Marchesini, 2003).
Proteoglycans (PG) in particular heparan sulfate
proteoglycans (H.S-PG) play an important role in the control of
charge selectivity in the glomerular capillary wall, being an
important component of the glomerular basement membrane
(GBM) (DeMuro, et at., 2001).
Urinary GAG and heparan sulfate are considered to be
markers of early renal involvement (Ilhan, et al., 2003).
٢

Introduction & Aim of the Work
Aim of The Work
To evaluate glycosaminoglycans (GAG), (HS) and CS
Levels in urine of systemic lupus erythematosus patients with
and without renal involvement and its role as a marker for lupus
nephritis with its correlation to disease activity.
٣

Review of Literature
Systemic Lupus Erythematosus
Systemic lupus Erythematosus (SLE) is an autoimmune
disease characterized by production of antibodies to components
of the cell nucleus in association with a spectrum of clinical
manifestations and a variable course characterized by
exacerbations and remissions (Salmon and Kimberly, 2000).
Clinical manifestations may be constitutional or result
from inflammation in various organ systems including skin and
mucous membranes, joints, brain, serous membranes, lung, heart
and occasionally gastrointestinal tract. Organ system may be
involved singly or in any combination involvement of vital
organs particularly, the kidneys and central nervous system
account for significant morbidity and mortality (Gladman and
Urowitz, 1997) (table I).
4

Review of Literature
Table (I): Frequancy of various clinical manifestations at
presentation or at any time during the course of SLE. Described
by (Gladman and Urowitz, 1997)
Manifestations At onset (%) At any time (%)
Constiutional 73 84
Arthritis 56 63
Arthralgia 77 85
Skin 57 81
Mucous membrane 18 54
Pleurisy 23 37
Lung 9 17
Pericarditis 20 29
Myocarditis 1 4
Raynaud’s phenomenon 33 58
Thrombophlebitis 2 8
Vasculitis 10 37
Renal 44 77
Nephrotic syndrome 5 11
Azotemia 3 8
C.N.S 24 54
Cytoid bodies 22 47
Pancreatitis 1 4
Lymphadenopathy 25 32
Myositis 7 5
5

Review of Literature
Epidemiology:
Sex status is obviously of great importance in
susceptibility to SLE, which is predominantly a disease of
women, particularly during their reproductive years (Lahita,
1999).
Peak incidence occurs between the ages of 15 and 40
during the childbearing years with a female to male ratio of
approximately 10:1, SLE affects approximately 1 in 200
individuals although the prevalence varies with race, ethnicity
and socioeconomic status (Ward et al., 1995).
Within the united states, during periods of relatively stable
populations dynamics of San Francisco in the Late 1960,
prevelance for white women was found to be 90. 5 per 100,000
(Fessel, 1974).
A more recent estimate from the united States used
random digit dialing to 16,607 house holds and from this
derived a sample of 4034 women older than 18 years, Cases that
were reported and validated through medical Chart review
provide a prevalence estimate of 124 per 100,000 adult women
(Hoch berg, et al., 1995)
6

Review of Literature
Etiology:
Several factors are included as etiologic factors for SLE
which include, genetic factors, diet, hormonal factors, drugs,
infections and ultraviolet Light.
1-Genetic factors:
Several lines of evidence indicate that genetic factors are
critical in the development of SLE supported by family studies.
Family members of SLE are more likely to have lupus or
another connective tissue disease. There is an eight fold relative
risk for SLE in first or second degree relatives (Deapen, et al,
1992). Another evidence of genetic predisposition is the
presence of a 3-to-l0 fold increase in clinical disease in
monozygotic compared with dizygotic twins (Jarvinen et al.,
1992). Lupus and lupus like syndrome are associated with
inherited abnormalities of the major histocompitability complex
(MHC) class III genes for complement but also deficiencies of
SLE associated with MHC class II (allo antigens) HLA. DR2
and HLA DR3 (Arnett, 1997).
DR3 and DR2 are individual genes that predispose to SLE
in multiple different ethnic groups but the relative risk conferred
by those genes alone is 2 to 3 (Arnett ,1997).
A few lupus patients also have genetic deficiencies of
complement components. Acquired complement deficiencies are
also associated with lupus (Stewart, 1999).
7

Review of Literature
In contrast to their uncertain role in disease susceptibility,
class II genes appear to exert a more descisive influence on the
production of particular ANA. The response to several SLE
autoantigens has been associated with particular class II alleles,
as well as short aminoacid sequences shared epitopes, may
influence antigen-specific responses by virtue of their location at
contact points of class II molecules with processed peptides
(Reveille et al., 1991).
2-Hormonal factor:
Menopausal women treated with hormone replacement
therapy are at increase risk for development of SLE compared
with that did not receive such therapy (Sanchez-Gurero et al.,
1998). And women exposed to estrogen containing oral
contraceptives also have increase risk for SLE (Sanchez.
Gurero et al.,1998).
3-Diet:
An additional factor that may promot the development of
SLE in genetically predisposed individuals is diet. Some
monkeys fed alfalfa sprouts developed SLE. Sprouting
vegetables contain an aromatic aminoacid L-canabarine, that is
immune stimulatory (Hahn, 2001).
8

Review of Literature
4-lnfection:
Viral infection has been suggested as an etiologic factor in
SLE, as infections may play a role in expanding undesirable
immune responses for examples, administration of bacterial
lipopolysaccharides to mice with SLE accelerates disease
(Cavallo and Grenholm; 1990). A study of children showed
that those with SLE were significantly more likely to have
evidence of infection with Epstein-Barr virus than were matched
non-SLE control children from the same population (Harley and
James, 1999). Investigations in one laboratory have implicated
human retrovirus in the synovitis of SLE and RA (Griffthis et
al.,1999).
5-Ultraviolet Light:
As many as 70 percent of SLE, have their disease flared-
by exposure to UV light (Wysenbeck et al., 1989). Mechanisms
by which UV light exposure accelerates the disease include
increase in the thymine dimers which render the keratinocytes to
UV light induces apoptosis exposing the self-molecules to the
immune system, or rendering them immunogenic (Casciola-
Rosen and Rosen, 1997).
9

Review of Literature
6-Drugs:
Drugs appear on the list of environmental exposures that
might induce SLE like disease. The most important drugs are
hydralazine, procainamide, isoniazide, hydantoins,
chlorpromazine, alfa methyledopa, D-penecillamine and
interferon. The clinical manifestations of drug induced lupus are
predominantly arthritis, serositis, fatigue, malaise and low grade
fever ; but nephritis .and central nervous system disease are rare.
These manifestations disappear in most patients .within few
weeks of discontinuation of the offending drug. Although all
patients have high titre antinuclear antibodies, it is unusual to
have high titre anti-DNA (Fritzler and Rubin, 1993)
Immuno pathogenesis:
Cellular immunity:
B cell activation is clearly abnormal in individuals with
SLE. B cell hyperactivity leads to hyperglobulinemia, increased
number of antibody producing cells and heightened responses to
many antigens, both self and foreign antigens (Klinman, 1997).
Role of T cell in the pathogenesis of SLE is found to be
critical. T cell function abnormalities are common in SLE. In all
the strains of lupus mice that have been tested, inactivation or
elimination of CD4 + helper T cell protect from the disease
(Wofsy.1986).
10

Review of Literature
Because many of the pathogenic auto antibodies in
patients with SLE are IgG, T cell help is necessary for their
production and maintainance. Auto antibodies production found
to be helped by cell of different surface phenotypes (Rajagoplan
et al., l990).
Direct tissue damage by cytotoxic T cell is suggested in
polymyositis associated with SLE or another connective tissue
disease (O, Hanlon et al., 1994). Also dermatitis in 50 precent
of individuals with subacute cutaneus lupus not having
immunoglobulin and complement deposited in dermo epidermal
junction, may be caused by T cells (Werth et al., 1997).
In human SLE , the total number of T-cells is usually
reduced, Propably owing to the effects of anti-Lymphocyte
antibodies (Horwitz, 1997)
There is evidence that SLE B cells are more easily
activated and driven to mature by cytokines stimulation than are
non- SLE B cells. For example, SLE B cells are more easily
driven to differentiate by IL-6 than are normal B cells (Honda
and Linker - Israeli, 1999)
Several experiments suggest that the normal events of T
cell activation, such as intracellular calcium increases, protein
kinase-A activation and generation of cyclic adenosine
monophosphate are defective in SLE –lymphocytes compared
with normal ones (Kammer, 1999)
11

Review of Literature
There is evidence that apoptosis is abnormal in T cells
from people with SLE (Lorenz et al., 1997).
Autoantigens in systemic lupus erethematosus are highly
diverse in terms of structure and location in control cells, but
become clustered in and on the surface blebs of apoptotic
cells.The past several years have provided significant evidence
that the apoptotic cell plays a central role in tolerizing B cells
and T cells to both tissue-specific and ubiquitously self-antigens
and may drive the autoimmune response in systemic
autoimmune disease. (White and Rosen, 2003)
Humeral immunity
SLE patients exhibit hyperactivity by humeral component
of immunlogic responsiveness manifested by hypergamma-
gloubulinemia, autoantibodies and circulating immune
complexes. The central immunologic disturbance in SLE
patients is autoantibody production. These antibodies are against
a host of self molecules found in the nucleus and cytoplasm in
patient’s sera, those directed against component of the cell
nucleus (Antinuclear antibodies or ANA) are the most
characteristic of SLE and are found in more than 95% of
patients. These antibodies bind DNA, RNA, nuclear proteins
and protein nucleic acid complexes.(Tan,1989)
12

Review of Literature
Among ANA specificities in SLE, two appear unique to
this disease antibodies to double stranded (ds) DNA and RNA-
Protein complex termed Sm. The IgG anti-double stranded DNA
response correlates with disease activity and nephritis in some
patients with SLE and some nephritogenic monoclonal
antibodies to DNA bind DNA-histone complex; the anti DNA
/DNA histone complex can then bind to heparan sulphate in
glomerular basement membrance (Ohnishi et al., 1994).
The relationship between levels of anti DNA and active
renal disease is not invariable, however; some patients with
active nephritis may lack serum anti DNA, where others with
high levels of anti DNA are clinically free and escape nephritis
(Pisetsky, 1992).
Auto antibodies in systemic lupus erythematosus:
Autoantibodies with multiple different specificities form
the immune deposits in glomeruli of patients with SLE,
including antibodies to dsDNA, Sm, SSA, SSB, the collagen
like region of CIq, and chromatin. (Mannik M, et al., 2003).
1 -Antinuclear antibodies (ANAs):
95% to 99% of patients with SLE demonostrate elevated
levels of anti-ANAs, which are considered to be the hallmark of
the disease but it is not specific for SLE (Brain and Kotzin,
1997). There are many conditions associated with a positive
ANA as seen in table (II).
13

Review of Literature
Table (II): The different conditions with positive ANA
Condition % ANA Positive
- SLE 95-99
- Healthy relatives of SLE patients 15-25
- Rheumatoid arthritis 50-75
- Mixed connective tissue disease 95-100
- Progressive systemic sclerosis 95
- Polymyositis 80
- Sjogren’s syndrome 75-90
- Cirrhosis (all causes) 15
- Auto immune liver disease (autoimmune
hepatitis, Iry billiary cirrhosis)
14
60-90
- Normal person 3-5
-Normal elderly (>70 ys) 20-40
- Neoplasia 15-25
(Emlen, 1996)

Review of Literature
ANA patterns
ANA patterns refer to the patterns of nuclear fluorescence
observed under the fluorescence microscope. Certain patterns of
fluorescence are associated with certain diseases, although these
associations are not specific. (Table III).
Table (III): the ANA Patterns of different diseases.
ANA Pattern Disease
-Homogenous diffuse SLE, drug induced LE, other disease
- Rim (Peripheral) SLE, chronic active hepatitis
- Speckled
- Nucleolar Scleroderma
SLE, Mixed connective tissue disease,
sjogren’s, Scleroderma, other disease
- Centromere Limited Scleroderma (CREST)
15
(Fritzler, 1987)
Few patients (1-2%) with active, untreated SLE will have
negative ANA In addition a larger number of SLE patient (10-
15%) will become ANA negative with treatment or as their
disease become inactive. SLE patient with end stage renal
disease on dialysis frequently become ANA negative (40-50%)
(Emlen, 1996)

Review of Literature
The spectrum of ANA
It includes antibodies against a wide variety of nuclear
components of chromatin including histones, high morbidity
group (HMG) protein and various forms of DNA, also the
cytoplasmic or nucleolar RNP, components of the nucleolus, or
other cellular elements (Craft and Hordin, 1993)
2)- Anti- DNA
Antibodies to DNA were first reported in 1959. the genetic
information of all living organisms are contained in deoxy
ribonucleic acid (DNA), three forms of DNA are antigenic in
systemic lupus erythematosus; single stranded (ssDNA), ,double
stranded (ds DNA) and left handed (Z DNA). Many lupus sera
contain antibodies that bind to more than one form of DNA.
However anti ds DNA antibodies are rarely found in diseases
other than SLE. Antibodies to ds DNA are detected in
approximately 60% of patient with SLE, and in general anti ds
DNA levels reflect disease activity (Pisetsky, 1992).
Anti ds DNA is highly specific in SLE but low titres may
appear in normal persons and patient with Sjogren's syndrome
and rheumatoid arthritis.
Also anti ssDNA may appear in drug induced lupus,
chronic active hepatitis and infectious mononucleosis. (Craft
and Hordin, 1993).
16

Review of Literature
Many anti-DNA antibodies are polyspecific and interact
with molecules other than DNA such as heparan sulphate and
laminin which are two glomerular constituents. The binding of
anti-DNA to these molecules could directly activate
complement to induce local inflammatory damage, this binding
could also fix immune complexes to kidney sites whether they
are formed in the circulation or in-situ (Pisetsky,. 1997)
3)-Anti- Sm and Ul RNP:
The sm antigen which is designated as sm RNP (small
nuclear ribonucleoprotein) is composed of unique set of uridine
rich RNA molecules (ul,u2, u3, u4, u5, u6) bound to a common
group of core proteins as well as unique proteins, specifically
associated with the RNA molecules, Anti sm -antibodies were
found to have no association between it and renal, neuro
psychiatric and cardiopulmonary features, but associated with
immune complex mediated vasculitis (Singh et al., 1991).
4) Anti Ro (SS-A) and Anti La (SS-B) antibodies:
Anti-Ro (SS-A) and anti-La (SS-B) antibodies
determination have become important serologic tests in the
evaluation of lupus erythematosus and sjogren’s syndrome
patients. These antibodies appear to identify a group of lupus
patients with prominent skin diseases (Provost, 1991).
17

Review of Literature
Anti-Ro antibodies are associated with a syndrome called
"neonatal lupus" which is secondary to the transport of maternal
IgG auto antibodies across the placenta after the first trimester
resulting in complete heart block that is typically irreversible as
well as transient skin rash, liver disease, and heamatologic
abonormalities in the neonate. (Buyon and Winchester, 1990).
Other autoantibodies in SLE:
1- Anti-lymphocytc antibodies :
lymphocytotoxic antibodies are demonstrable in the serum
of at least 80 precent of patient with SLE. (Winfield and
Mimura, 1992). Defects in T-suppressor functions are one of
the most constant findings in SLE, it is of considerable interst
that SLE anti-lymphocyte antibodies may show specific reactive
for an ability to inactivate or kill supperessor T-lymphocytes or,
inhibit IL-2 production by lymphocytes (Tanaka et al., 1989).
2- Rheumatoid factor and cryoglobulinaemia
Patients with SLE who are rheumatoid factor (RF)
negative, cryoglobulin positive are likely to develop renal
disease, where as those who are RF positive and cryoglobulin
negative are very unlikely to occur. (Howard et al., 1991).
18

Review of Literature
3- Anti cardiolipin antibodies
These antibodies have assumed importance in the past
decade because their presence has been associated with fetal
loss, particularly in late pregnancy (Harris et al., 1985). This is
probably due to thrombosis occurring in the placental vessels.
(Ramsy - Goldman et al., 1992).
Travkina et al., (1992) found relationship between the
development of certain neurological disorders (chorea, cerebral
circulation impairment, convulsive syndrome) and cardiolipin
antibodies in SLE patients.
4- The lupus anticoagulant (LAC)
The effect of antiphospholipid (apl) antibodies are
multifold. Affecting both humeral and celluar components
involved in hemostasis apls typically need to interact with
certain plasma proteins in order to react with negatively charged
phospholipid (Nimpf, et al., 1986).
Patients with SLE and APS have antibodies directed
against high density lipoproteins (HDL) and apolipoprtein A-l
(Apo A-l), high percentage of these antibodies cross react with
anti- cardiolipin (CL) suggesting the presence of different
groups of antibodies with different targets (Delgado, etal 2003 )
19

Review of Literature
Antiphospholipid (anticardiolipin) antibodies can be
detected either in patients with systemic auto immune
syndromes such as systemic lupus erythematosus or as isolated
phenomena in the so called primary antiphospholipid syndrom
(APS). The laboratory hallmark of this syndrom is a positive test
for lupus anticoagulant, the presence of anticardiolipin
antibodies or both (Greaves, 1997)
The presence of these antibodies is associated with
pathological changes in the microvasculature of many organs
and it is possible that the antibodies themselves are pathogenic
(Griffiths and Papadakil Neild GH., 2000).
20

Review of Literature
Immunopathology
There are several characteristic lesions in the pathology of
SLE which do not present in other diseases and these lesions
include haematoxylin bodies, onion skin lesion, libman sacks
endocarditis.
Heamatoxylin bodies:
Heamatoxylin bodies are usually oval or spindle shaped
basophilic structures which may approach the size of intact cell.
It is also known as "LE bodies”. Heamatoxylin bodies resemble
aggregates of chromatin and degenerated cytoplasmic organelles
and can be formed in vitro with epithelial cell nuclei. (Cruik
shank, 1987).
These bodies are most often described in glomeruli and
endocardium and can be found in almost any organ. Engulfment
of heamatoxylin bodies by phagocytes produces the
characterestic "LE cell" (Edberg et al, 1994).
Onion skin lesions:
Are microscopic vascular findings appear as concentric
arterial fibrosis, typically seen in the malpigian corpuscles of the
spleen. The laminated arterial lumen assumes an onion skin
appearance presumably reflecting the end stage of arteritis
similar lesions can also be seen in thrombotic thrombocytopenic
purpura. (Edberg et at, 1994).
21

Review of Literature
Libman Sacks endocarditis:
The majority of SLE patients at autopsy have non
bacterial, verrucus endocarditis known as Libman Sacks
endocarditis. Grossly these small friable vegetations are often
present in large numbers. The mitral valve is the most
commonly affected. Microscopically, the verrucae consist of
proteinaceous deposits and mononuclear cells in the context of
platelet thrombi and necrotic cell debris (Pisetsky., 1993).
The pathology of the disease in the skin, vessels, kidney
I-cutaneus immuno pathology:
Immune complex formation with consequent tissue
damage, acute vascular and perivascular inflammation and
mononuclear cell infiltration are the basic pathoplysiologic
mechanisms in SLE. Each of these features can be found in the
histopathology of cutaneus lupus although immunoglobulin
deposition at the dermo-epidermal junction is the most
consistent.
In a majority of patients with systemic disease, immunofluorscence,
reveals similar deposits at the dermo-epidermal
junction in non sun exposed skin as well as at site of cutaneus
lesions.This characterestic immunofluorescence, (the lupus band
test) may be helpful in diagnosis although it can be also positive
in RA, sjogren’s syndrom, dermatomyositis, progressive
systemic sclerosis, and other clinical conditions (Douglas smith
et al., 1984).
22

Review of Literature
II-Vascular immunopathology:
SLE is characterized by wide spread vasculitis affecting
capillaries, arterioles and venules (Huskisson, 1999). The first
lesions are characterized by granulocytic infiltration and
periarteriolar oedema. This is followed by round cell infiltration
and a cellular oesinoplilic material composed of fibrin,
immunoglobulins and complement containing scattered
herematoxylin bodies (Steinherg 1992).
Ill-Renal immunopathology :
WHO classify the different pathologic forms of lupus
nephritis as 6 classes (Kotzin et al., 1996) (Table III)
a-Normal b-Mesangial nephritis
c- Focal prolifrtative
d- Diffues proliferative
e- Membranous nephropathy
f- Sclerosing
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Table (IV): Renal immunopathology
Class of patient Renal histology Clinical presentation prognosis
I- Normal Normal No abnormalities Good
II-Mesangial -Mesangial hypertrophy Upto 25% no abnormal- Good
-Mesangial immune lities-transient minimal
deposits
protienuria and /or
haematuria ↓C3, C4 and
↑anti DNA in one third
III-Focal
-Both mesangial Mild proteinuria Moderate
proliferative endothelial proliferation (

Review of Literature
Pathogenesis:
Lupus Nephritis
“Lupus nephritis is a classic example of immune complex
glomerulonephritis. Deposits may be found in all three
glomerular regions (subendothelial, subepithelial and mesangial)
(Adler, et al., 1995).
The chronic deposition of circulating immune complexes
may play role in certain patterns of glomerulonephritis such as
the mesangial and endocapillary proliferafive patterns. The
localization of circulating immune complexes within the
glomerulus is influenced by the size, charge and avidity of the
complexes, the clearing ability of the mesangium, and all local
hemodynamic factors. Once these immune complexes are
deposited, the complement cascade is activated, leading to
complement mediated damage, activation factors, leucocyte
infiltration release of proteolytic enzymes, and various cytokines
regulating glomerular cell proliferation and matrix synthesis
(Appel and D’Ag et al., 1995).
Several studies have suggested that binding of DNA to
glomerular basement membrane (GBM) follwed by in situ
reaction with anti DNA antibody may participate in the genesis
of the glomerular lesions of SLE in experimental models. In
vitro binding of circulating imuune complex to GBM also
occurs (Adler et al., 1995). In SLE, nephritis has been most
25

Review of Literature
intensively studied because of the impact of kidney disease on
morbidity and mortality. Clinical observation strongly suggest
that SLE renal disease results from the deposition of immune
complexes containing anti-DNA. Since active nephritis is
marked by elevated anti-DNA levels with a corresponding
depression of total haemolytic complement. Furthermore, anti
DNA shows preferential renal deposition and these findings
suggest that DNA anti DNA immune complexes are major
pathogenic species (West et al., l995).
Although renal injury in SLE may result from immune
complexes that contain anti DNA, the constituents of circulating
complexes have been difficult to characterize because of their
low concentration in serum. The formation of immune
complexes insitue rather than within the circulation could
explain the paucity of DNA anti-DNA complexes in serum.
According to this mechanism, immune complexes would be
assembled in kidney on pieces of DNA adherent to the
glomerular basement membrane. These DNA pieces may be
attached to histones which also bind strongly to glomerular sites
(Pisetsky, 1997).
Transition between better and worse of renal involvement
have been quantified with respect to the likelihood they would
progress or improve (Edworthy , 2000).
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Review of Literature
The individuals who exhibit normal kidney function are
likely to retain normal function during the coming years if they
have serum albumin levels above 40 mg/L and normal systolic
blood pressure at their annual check up. However, if serum
albumin is low and lymphocyte counts are less that 1000, the
likelihood of progression is at least 50 percent, Approximately
25% of patients found to have proteinuria but without evidence
of azotemia will progress to azotemia during the next 10 to 12
months, particularly those with a combination of excess red
blood cells on urine analysis and either low white cell count or
low complement levels. Once kidney function has decreased
sufficiently to produce a serum creatinine level more than 400
mg/L, most of patients will require dialysis or kidney
transplantation within a year. Some studies have suggested
patients who progress to renal failure will have immunologic
disease activity subsequently (Edworthy, 2000).
Along with these parameters, the level of serum albumin
and cholesterol will provide markers for the nephritic syndrome
as will as the severity of the proteinuria. Worsened proteinuria is
an indicator of poor outcomes with respect to renal function
(Fraenkel et al., 1999).
The pathologic findings have been correlated with clinical
and laboratory findings and are often used to suggest renal
prognosis in individual patients.
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Review of Literature
In general:
Class I: Normal findings on biopsy is thought to have an
excellent prognosis.
Class II: Mesangial hypertrophy and mesangial immune
deposits generally have a good prognosis.
Class III: Mesangial and endothelial proliferation with immune
deposits along capillaries but less than 50% of
glomeruli involved, have a moderate prognosis.
Class IV: Diffuse proliferative glomerulonephritis with greater
than 50% of glomeruli involved and cell proliferation
resulting in crescent formation, these patients will have
poor prognosis but may be amenable to aggressive
immunosuppressive therapy.
Class V: Membranous glomerulonephritis with subepithelial
granular immune deposits is associated with nephritic
range proteinuria in two thirds of patients, but patients
often maintain normal creatinin clearance.
Class VI: Sclerosing changes with fibrous crescents and
vascular sclerosis, is ominous sign that there are few
reversible elements to the kidney involvement and that
progression to renal failure is likely.
The clinical and laboratory associations with class II to
class V are provided in the next table (Edworthy, 2000).
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Table (V) Correlation between clinical and laboratory findings and
pathologic classifications in lupus nephritis patients.
Mesangial Focal
proliferative
IIA IIB
III
Diffuse
proliferatice
IV
Symptoms None None None Renal failure
29
Membranous
V
Nephritic
syndrome
Hypertension None None ± Common Late onset Late onset
Tubulointerstitial Infection
None Dysuria
Late
onset
Proteinuria None < 1 < 2 1-20 3.5-20 ± ± ±
Heamaturia
(RBC’s/HPF)
Pyuria
(WBC’s/HPF)
Drug
induced
Renal
failure
None
None 5-15 5-15 Many None ± ± None
None 5-15 5-15 Many None ± Many None
Casts None ± ± None None None None None
GFR
(ml/min)
NI NI 60-80 < 60 NI NI NI NI or ↓
CH50 NI ± ↓ ↓↓ NI NI NI NI
C3 NI ± ↓ ↓↓ NI NI NI NI
Anti DNA NI ± ↑ ↑↑ NI NI NI NI
Immune
complexes
NI ± ↑ ↑↑ NI NI NI NI
C3: complement 3 GFR: Glomerular filtration rate
HPF: High power field I: occasional or small amount
NI: Normal
CHS: Total serum heamolytic complement (expressed in 50% heamolytic
units)

Review of Literature
Mortality and Morbidity:
Morbidity is related to the renal disease itself, as well as
treatment related complications and co-morbidities, including
cardiovascular disease and thrombotic events. Progressive renal
failure leads to anemia, uremia, and electrolyte and acid base
abnormalities. Hypertension may lead to an increased risk of
coronary artery disease and stroke. Nephritic syndrome may
lead to oedema, ascitis and hyperlipidemia, which add to the risk
of coronary artery disease and thrombotic tendency
(Kashgarian, 1997).
There are general manifestations as patients with active
lupus nephritis often complain of fatigue, fever, rash, arthritis
and serositis, these are more common with focal proliferative
and diffuse proliferative lupus nephritis. As regards renal
manifestations, symptoms related to active nephritis may
include peripheral oedema secondary to hypertension or
hypoalbuminemia. Extreme peripheral oedema is more common
in diffuse proliferative and membranous lupus nephritis
(Lawrence, 2002).
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Proposed immuno pathogenic mechanisms of Lupus
nephritis:
There are three basic hypothetical mechanisms by which
autoantibodies can cause nephritis. The circulating immune
complex hypothesis, the cross-reactive antibody hypothesis and
the planted antigen hypothesis. Table (VI) (James and Gary,
1996)
Table (VI): Proposed immuno pathogenic mechanisms in
Mechanism Comments
Circulating
immune
complexes
Crossreactive
antibodies
Planted
antigen
lupus nephritis.
-Nucleosome / antinucleosome complexes are likely to
participate in nephritis
-Existence and role of DNA/Anti DNA coplexes is controversial.
- Cryglobulins may also contribute to nephritis
- Role of other immune complexes in nephritis is uncertain.
- Variant of molecular mimicry them
- Recent technical and theoretical concerns warrant reappraisal
of polyreactivity concept
- Propobly less common than binding of auto antibodies to their
cognate antigen
- Histones and nudeosomes bind avidly to glomerular basement
membrane (GBM) and glomeruli -Histones Facilitate the
binding of DMA to GBM
- Type IV collagen and heparan sulfated Proteoglycans are
important for histone /nucleosome binding to GBM
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Review of Literature
Nephritogenic autoantibodies in Lupus
1) Auto antibodies to CIq:
Autoantibodies against CIq can be found in the circulation
of patients with several autoimmune diseases including systemic
lupus erythematosus (SLE). In SLE there is an association
between the occurrence of these antibodies and renal
involvement. (Trouw LA, et al., 2004).
Although the mechanism by which arti- CIq antibodies
contribute to nephritis is uncertain, one possibility is that they
act to amplify the inflammatory response by binding to CIq that
has bound to immune complexes within the glomerulus
(Uwotoko , et al., 1997)
New assays ( anti -CIq and anti nucleosome antibodies)
have been recently proposed for diagnosis and monitoring SLE
patients with promising results (Sinico, et al., 2002) .
Nucleosoms are a major auto antigen in SLE, Besides
serving as an immunogen for the pothogenic Tcells and Bcells,
nucleosomes contribute to the development of lupus nephritis by
mediating the binding of antinuclear artibodies to GBM (Table
VII) (Shoenfield 1994)
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Review of Literature
Table (VII): Additional evidence for the In Vivo Relevance
of Nucleosome- Mediated Binding of Autoantibodies to
the Glomerular Basement membrane In SLE Nephritis:
- Elution of antibodies from glomeruli disclosed specifities
toward all components of the nucleosome, such as DNA,
histones, and nucleosoms
- Histones and nucleosomes are present in glomerular
deposits in human SLE .
- Anti - heparan sulfate reactivity was elevated as a method
to identify nucleosome complexed auto antibodies in the
circulation at the onset or during exacerbation of SLE
nephritis.
- Heparin and non coagulant heparin derivatives inhibited
the binding of neucleosome -complexed autoantibodies to
the GBM in the in vivo renal perfusion.
2-Anti - Endothelial cell Antibodies (AECA ):
Sera from patients with systemic lupus erythematosus have
been reported to contain IgM and/or IgG binding to endothelial
cells (EC), i.e. anti-EC antibodies (AECA). (Renaudineau et
al., 2002).
There is aconsensus on the high prevelance of AECA in
SLE. There is an association between the AECA and renal
involvement of SLE. Several groups have now confrirmed and
extended the correlation between SLE disease activity and the
presence of AECA (D’cruz, et al.,1991)
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Review of Literature
Assessment of SLE activity
There are various approaches to the measurements Of
disease activity in SLE patients, these include the assessment of
clinical features, the monitoring of certain laboratory tests,, or
various combinations of the two (Hay and Emery, 1993).
Activity indices of SLE:
From 1960 to the present, over 60 different disease activity
indices have been described. Although non of them is perfect.
Most indices are reliable, valid and suitable for classifying and
monitoring groups of patients in the research settings. (Hay and
Emery, 1993).
Many indices are based on clinical findings alone, without
incorporating treatment. They provide a score related to the
extent of inflammation involved. The British Isles Lupus
Assessment Group score incorporate treatment decisions into
the assessment. The UCH /Middel sex score incorporates the
dose of steroids plus other clinical findings. Incorporating
therapy is an indication of the clinicians overall impression of
activity (Gladman, 1994).
The weights given to each variable do not always
correspond to the degree of inflammation present, instead, they
may relate to the seriousness of the organ involvement
according to the life threatening nature or threat to the functional
capacity of the individuals. For example the weights given by
34

Review of Literature
the SLE Disease Activity Index are much greater for CNs
involvement than they 'are for cutaneous involvement. In this
score retinal haemorrhages or optic neuritis are given 8 points
while discoid patch or skin involvement is given 2 (Bombardier
et al., 1992).
The Ropes system, described an evaluation scheme based
on (clinical and glaboratory features for rating activity, five of
the laboratory variables decribe renal manifestations (Liang, et
al., 1989). The British Isles Lupus Assesssent Groupe Scale,
(BILAG) consists of 109 unweighted items. these are scored as
being abscent, present, active, progressive or recurring within 3
months (Bacon, et al, 1986).
The SLAM, covers symptoms that occurred during the
previous month, and includes 24 clinical manifestations and
laboratory parameters to evaluate organs which cannot be
assessed otherwise, parameters of immune functions are not
included (Liang et al, 1989)
Laboratory markers of disease activity in SLE:
(1) Erythrocytic sedimentation rate and C. reactive protein:
The ESR is frequently elevated in active SLE, but it dose
not mirror lupus activity and may remain elevated in patients
with prolonged clinical remissions (Gladman and Urowitz,
1997).
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Review of Literature
While CRP levels are elevated above the normal range in
most patients with active SLE and tends to fall and rise as the
disease improves or becomes more active, a number of patients
with active SLE do not show even mild CRP elevation (Ballow
and Kushner, 1997).
(2) Raised titre of antibodies to ds DNA:
Several reports have claimed a corelation between disease
activity and levels of anti-ds DNA antibodies in SLE (Nossent
et al., 1989) especially with renal involvement (Ter Borg et al.,
1990).
The relationship between IgA anti-ds DNA antibodies and
SLE disease activity was investigated and concluded that their
increased levels correlate with disease activity. The presence of
IgA anti-ds DNA antibodies associated with kidney, joint
abnormalities, hypocomplementemia and with circulating
immune complexes (Miltenberg, 1993).
The auto autibodies most closely associated with lupus
nephritis are anti-DNAantibodies. Anti -DNAantibodies are a
principal feature of both murine and human SLE, and their
presence correlates with nephritis (James and Gary, 1996)
(3)complement component and disease activity:
Measurment of complement 3 (C3) and /or complement 4
(C4) are less sensitive than anti ds-DNA antibodies in predicting
an exacerbation in SLE (Ter-Borg et al., 1990).
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Review of Literature
Petri et al., (1991) concluded that the reduction of the
levels of C3 and C4 complement components frequently
characterizes SLE flare. (Rother, 1993) ,found that complement
split produces (C3 d) and C3 d/C3 provide sensitive marker for
disease activity in SLE, since C3 d is a direct measurment of
complement turn over, it reflects complement activation better
than C3 , C4 and CH50
Hypocomplementemia is found at presentation in more
than three quarters of- untreated patients with lupus, and ismore
common with evident nephritis. The concentration of C4 and clq
tends to be more depressed than C3, which suggests
complement activation via the classical pathway. So,
complement- based tests for “immune –complexes” have been
shown to measure not immune complexes but anti-clq
autoantibodies (Stewart, 1999)
Anti -ds DNA antibodies are highly specific for SLE and
present in a hiqh proportion of SLE patients (40- 80 % )
“Sinico, et al., 2002”
(4) Soluble tumour necrosis factor receptor (sTNFR):
There is acorrelation between serum levels of soluble
tumour necrosis factor receptor and disease activity in SLE. An
increase in serum levels of sTNFR is a useful marker for SLE
activity where it shows stronger correlation than do any other
laboratory or clinical parameters (Aderka, 1993).
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Review of Literature
(5) Leukotrein E4 (LTE4):
The peptido leukotreins including leukotrein E4 and its
metabolites LTD4 and LTE4 are lipoxygenase products of
arachidonic acid. The execretion of LTE4 as an index of total
body peptido leukoterin production levels is increased in
patients with SLE and the urinary levels were found to correlate
with clinical manifestation of disease activity using the SLAM
index in comparison with the ESR and /or anti-ds DNA. More
over, Urine LTE4 may be the early recognition of disease flare
and treatment failure (Maltby et al., 1990).
(6)Anti-U1 RNP antibodies:
This was studied in SLE patients with overlap syndrome
and their level can be useful for monitoring disease activity
(Hoet et at., 1992).
(7) Serum cystidine deaminase (CD):
Cystidine deaminase (CD) is a substance that has
undergone limited evaluation as disease activity marker. It is a
cytoplasmic enzyme produced during nucleic acid breakdown.
CD is distributed in various body tissues. With the heighest
levels of activity in solid organs found in the liver, placenta,
lung and kidney. High concentrations of CD are also found in
mature neutrophils with level many times greater than in other
blood elements such as lymphocytes.
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Review of Literature
However, there was a significant correlation found
between CD levels and anti-DNA antibody titre as well as serum
C3, more over a subgroup of patients with active SLE has been
identified with abnormal CD levels but normal anti-DNA titres
and was characterized by arthritis in the majority of patients.
This suggests that, at least in certain clinical situations Of SLE,
CD determination may provide more useful information for
evaluation and monitoring of these patients than anti DNA
antibody litres, but longer term prospective analysis are required
(Lamberts, 1994).
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Review of Literature
Glycosaminoglycans
(GAGS)
Glycosaminoglycans (GAGS; Mucopolysaccharides) are
long, highly negatively charged, unbranched polymers of
repeating disccharide unites.
Although six distinct classes of glycosaminoglycans are
now recognized Hyaluronan, Chondriotin sulfate, Heparin,
Heparan sulfate, Dermatan sulfate and Keratan sulfate, certain
features are common to all classes. The glycosaminoglycans’
side chains found on a particular core protein are commonly
divided into three basic groups; chondriotines, heparines, and
keratan sulfates (Wight, et al., 1991).
The carboxyle and sulfate groups contribute to the nature
of glycosaminoglycans as highly charged polyanions. Their
biological roles are lubricants and support elements in
connective tissue (Schwarts and Smalheiser, 1989).
The proteoglycans are a diverse group of uniquely
glycosylated proteins that are ubiquitous in the body and most
abundant in the extracelular matrix of connective tissues, like
glycoproteins, proteoglycans often contain both “N-linked and
o-linked oligo saccharides, however, the addition of one or more
sulfated glycoaminoglycans side chains to the protein core
distinguishs this group complex of glycoconjugates as
proteoglycans (Kuettner, 1994).
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Review of Literature
Figure (1): Structure of a proteoglycan. [Reproduced with
permission from J. E. Silbert and G. Sugumaran, Intracellular
membranes in the synthesis, transport,. and metabolism of
proteoglycans. Biochim. Biophys. Acta 1241: 372(1995).
41

Review of Literature
Biosynthesis of chondriotin sulfate is typical of
glycosaminoglycan formation:
The formation of the core protein of the chondriotin sulfate
proteoglycan is the first step in this process. The polysaccharide
chains are assembled by six different glycosyltransferase.
Polymerization then results from the concentrated action of two
glycosyltransferases, an N-acetylgalactosaminyltransferase and
a glucuronosyltransferase, which alternately add the two
monosaccharides forming the characteristic repeating
disaccharide units. Salfation of N-acetylgalactosamine residues
in either the 4or 6 position apparently occurs along with chain
elongation. The sulfate donor in these reactions as in other
biological systems is 3- phosphoadenosine-5phosphosulfate
(PAPS) which is formed from ATP and sulfate. (lennnarz,
1980).
Synthesis of other glycosaminoglycans requires additional
transferases specific for the sugars and linkages found in these
molecules. Completion of these glycosaminoglycans often
involves modifications in addition to 0-sulfation, including
epimerization. acetylation and N-sulfation (Devlin, 1992).
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Review of Literature
Figure (II): Synthesis of chondroitin sulfate proteoglycan.
Abbreviations: Xyl, xylose;Gal, galactose; GlcUA, glucuronic
acid; GalNAc, N-acetylgalactosamine; PAPS; phosphoad-
enosinephosphosulfate.
Regulation of the degree and position of sulfation may be
important for function. Indeed, two diseases of proteoglycans
synthesis, the diastrophic dystrofy type of
osteochondrodysplasia and macular corneal dystrophy type I
both result from the absence of sulfate groups on proteoglycns
(Bhagavan, 2002).
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Review of Literature
Hyaluronic acid (HA)
Figure (III): Repeat unit of hyaluronic acid from(Lidholt,
1997)
Hyaluronic acid is unsulfated, not covalently complexed
with protein and is the only glycosaminoglycan not limited to
animal tissue but is also produced by bacteria.
It consists solely of repeating disaccharide unites of N-
acetyle glucosamine and glucuronic acid. Although hyaluronate
has the least complex chemical structure of all the glycosaming-
lycan. The chains may reach molecular weights of 10 5 –10 7 .
The large molecular weight, polyelectrolyte character, and
large volume of water it occupies in solution, all contribute to
the properties of hyaluronic acid as a lubricant and shock
absorbant hence it is found predominantly in synovial fluid,
vitrous humor, and umbilical cord (Devlin, 1992).
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Review of Literature
Juha etal 2001, stated that hyaluronic acid is a critical
GAG in synovial joints, predominent GAG in the articular
surface and also is a key component of synovial fluid
The exact connection between GAG and oxidation stress is
not entirely clear, it is known that the end-end surfaces of bones
where they meet in a joint require ample and intact prescence of
hyaluronic acid, also it is known that HA can be damaged by
reactive oxygen molecules, these molecules multibly when parts
of the body are temporarly deprived of oxygen “a process
called, hypoxia – reperfusion” (Wool et al., 1992).
Hyaluronic acid may be important in permitting cells to
migrate through the extracellular matrix. Tumour cells can
induce fibroblasts to synthesize greatly increased amounts of
this GAG, thereby perhaps facilitating their own spread, some
tumour cells have less heparan sulfate at their surfaces and this
may play a role in the lack of adhesiveness that these cells
display (Robert, et al., 2000).
Extracellular catabolism of hyaluronan has never been
demonstrated in cartilage or other tissues, however
accumulating evidence suggests that turnover is primarily
intracellular following endocytosis by hylauronan receptors such
as CD44 (Chow et al ,1995).
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Review of Literature
Chondriotin sulfate (CS)
Figure (IV): The repeating disaccharide units of (a)
chondroitin 4- sulphate and (b) chondoritin 6- sulphate. From
(Lidholt, 1997).
The most abundant glycosaminoglycans in the body are
the chandriotin sulfates, the individual polysaccharide chains are
attached to specific serine residues in a protein core of variable
molecular weight through a tetrasaccharide linkage region. The
disaccharide may be sulfated in either the 4 or 6 position of N-
acetylegalactosamine.
Each polysaccharide chain contains between 30 and 50
sugars, such disaccharide units corresponding to molecular
weihts of 15.000-25,000.
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Review of Literature
Chondriotin sulfate proteoglycans have also been shwon to
aggregat non covalently with hyaluronate, forming much larger
structures. They appear to exist in vivo in this aggregated form
in the ground-substance of cartilage and have also been isolated
from tendons, ligaments and aorta (Devlin, 1992).
There has been found that, chondriotin sulfates are also
located inside certain neurons and may provide an endoskeletal
structure helping to maintain their shape (Hardingham and
Fosang, 1992).
Monoclonal antibodies 3-B-3 and 7-D-4 are specific for
chondriotin sulfate and recognize new epitopes which may
reflect pathophysiological events occurring in RA and
osteoarthritis (Caterson, et al., 1995).
Middleton, et al., 1999, found that 3-B-3 and 7-D-4 occur
in elevated levels in human articular cartilage from RA and OA
joints. Chondriotin sulfate proteoglycans (CSPG) is a major
constituent of the normal glomerular basement membrane(
GBM). (Pierina et al., 2002).
Medicinial preparations containing chondriotin sulfates
have been used as adjuvant in ocular surgery. CS extracted from
cartilage also have been used in degenerative articular diseases
because of their protective effects on cartilaginous tissues.
(Conrozier, 1998).
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Review of Literature
Heparin and Heparan sulfate (HS)
Figure (V): The anti-thrombin-binding pentasaccharide in
heparin/HS, [Reproduce with permission from K, Lidholt,
Biosynthesis of glycosaminoglycans in mammalian cells and in
bacteria. Biochem Soc. Trans. 25: 866 (1997).
Heparin is found in many connective tissues, it is not
present as a structural component but as an intracellular
component in most cells. It occurs in skin, lung, umbilical cord
and particularly in large amount in bovine liver capsule and pig
gastric mucosa.
The later two used as commercial sources of heparin.
Heparin contains glucosamine and uronic acid in its
disaccharide repeating unit and is highly sulfated.
A large proportion of glucosamine residues contain N-
sulfate group instead of N-acetyl groups. Both glucouronic acid
and iduronic acid are present in molecule (Murrary, 1988).
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Review of Literature
Heparan sulfate contains a similar disaccharide repeat unit
but has more N- acetyle groups, fewer N- sulfate groups., and a
lower degree of O-sulfate groups. Heparan sulfate appears to be
extra cellular in distribution and has been isolated from blood
vessel walls, amyloid and brain. It has been shown to be an
integral and ubiquitous component of the cell surface (Lennarz,
1980).
Proteoglycans and their glycosaminoglycans components
have effects on protein synthesis and intranuclear function,
heparin particularly seems to have an effect on chromatin
structure in vitro. It is not clear how physiologic these actions
are. Numerous storage or secretory granules such as the
chromatin granules in adrenal medulla, the prolactin secretory
granules in pituitary gland and the basophilic granules in mast
cells contain sulfated glycosaminoglycans.The
glycosaminoglycans’ peptide complexes that occur in these
granules may play a role in the release of biogenic amines
(Scott, 1992).
The more acute anticoagulation properties of the most
cells are met by different heparin-bearing proteoglycan,
serglycin, the core protein of which contains numerous serine /
glycin repeats within the central portion of the molecule
(Roughly and Pool, 1993).
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The presence of heparan sulfate proteoglycans on the
surface of vasculature lining endothelial cells may also serve to
provide other functions specific for heparine and heparan
sulfates such as binding to antithrombin III and binding to
lipoprotein lipase. (David, 1993).
The proteoglycan heparan sulfate perlecan is a “major
constituent of basement membrane (Bhagavan, 2002).
In addition to the well known anti coagulant effect of
heparin and heparnoids, some other pharmacologic properties of
this class of compounds have been described and applied to the
treatment of ischemic heart diseases, atherosclerosis and
hyperlipidemias. (Traini AM, et al., 1994).
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Review of Literature
Dermatan sulfate
Figure (VI): Repeat unit of dermatan sulfate from
(Lidholt, 1997).
Dermatan sulfate differs from chondriotin 4 and 6 sulfates
in that its predominant uronic acid is L - iduronic acid, although
D-glucuronic acid is also present in variable amounts.
"Schwartz and Smalheiser 1989"
Unlike the chondriotin sulfates, dermatan sulfate is
antithrombotic like heparin, but in contrast to heparin, it shows
only minimal whole blood anticoagulant and blood lipid -
clearing activities. As a connective tissue macromolecule,
dermatan sulfate is found in skin, blood vessels and heart valves.
(Devlin, 1992).
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Review of Literature
Keratan sulfate
Figure (VII): Repeat unit of Keratan Sulfate from (Lidholt,
1997).
More than any of the other glycosaminoglycans, keratan
sulfate is characterized by molecular heterogenicity. This
polysaccharide is composed principally of a repeating
disaccharide unit of N- acetyleglucosamine and galactose, with
no uronic acid in the molecule.
Sulfate content is variable, with ester sulfate present on C-
6 of both galactose and hexosamine. Two types of keratan
sulfate have been distinguished, which differ in their overall
carbohydrate content and tissue distribution. Both contain an
additional monosaccharides, mannose, fructose, sialic acid and
N-acetylgalactosamine. (Shwartz and Smalheiser, 1989).
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Both keratan sulfate and dermatan sulfate are present in
the cornea, they lie between collagen fibrils and play a critical
role in corneal transparency. Changes in proteoglycans
composition found in corneal scars disappear when the cornea
heals. The presence of dermatan sulfate in the sclera may also
play a role in maintaining the overall shape of the eye. (Robert
et al., 2000).
Functional aspect of glycosaminoglycans
Particular functional attributes of proteoglycans may
reside in the glycosaminoglycans structure or in the core protein
At present, 20 separate genes have been identified that
code for proteins and have the capacity to carry one or more
glycosaminoglycan side chains, Table (VII). once the nucleic
sequence of a proteoglycan core protein gene product has been
described and documented, the core proteins are often-given
functional names such as aggrecan, decorin and lumican (Wight,
et al., 1991).
53

Review of Literature
Table (VIII): Summary of proteoglycan properties.
Proteoglycan Interacts Glycosaminoglycan Predominent tissue
with
localization
Aggrecan Hyaluronan CS \KS Load bearing
tissues e.g cartilage
, aorta, disc, tendon
Versican Hyaluronan CS Ubiquitous, fibrous
Decorin Collagen
type I & II
CS or DS Ubiquitous
Biglycan Collagen
type VI
CS or DS. Ubiquitous
Fibromodulin Collagen
types l&Il
KS Ubiquitous
Collagen IX Collagen
type II
CS Cartilage
Syndecan 1 Cell
membranes
HS/CS Epithelial cells
Syndecan2 Cell . HS/CS Epithelial cells
(fibroglycan) membranes
Syndecan 3 Cell HS/ CS Ubiquitous
(N-syndecan)
Syndecan4
(ryudocanor
amphiglycan)
membranes
Cell
membranes
HS/CS •Ubiquitous
Glypican Cell
membranes
via glyeosyl
phosphatidy
l inositol
HS Ubiquitous
Serglycin Heparin Mast cells
Perlecan Basement
membranes
HS Basement membranes
The binding between glycosaminoglycans and other extra
cellular macromolecules contribute significantly to the structural
organization of connective tissue matrix, glycosaminoglycans
can interact with extra cellular macromolecules plasma proteins,
54

Review of Literature
cell surfaces components and intracellular macromolecules.
Some proteoglycans appear to serve as receptors and carriers for
macromolecules, including the lipoprotein, lipase, and
antithrombin. Proteoglycan seem to be involved in the
regulation of cell growth, the mediation of cell communication
and the shedding- shielding of cell surface receptors (Scott,
1992).
The presence of glycosaminoglycans localized directly on
the cell surface provides cells with an equisite means for
controlling the local cell environment this may involve all
functions of glycosaminoglycans including regulation of local
hydration and molecular movement, binding of basic growth
factors and cytokines and binding to other matrix components as
well as adjacent cell surface heparan sulfate receptors as binding
to antithrombin III and binding to lipoprotein lipase (David,
1993).
In order to maintain electrical neutrality, negatively
charged (anionic) groups pf glycosaminoglycans, fixed to the
matrix of the connective tissue, are neutralized by positively
charged (cationic) groups as Na osmotic pressure within the
matrix is thereby elevated thus, proteoglycans, because they
contain glycosaminoglycans, are polyanionic and usually
occupy large hydrodynamic volumes relative to glycoproteins or
globular proteins of equivalent molecular mass
(Bhagavan, 2002).
55

Review of Literature
Hyaluronidase (prepared from mammalian tests) is used
therapeutically to enhance dispersion of drugs administerated in
various parts of the body (Bhagavan, 2002).
Deficiencies of enzymes that degrade glycosaminoglycans
result in mucopolysaccharidosis which are inherited in an
autosomal recessive manner, with Hurler’s and Hunter’s
syndromes being perhaps the most carefully studied specific
laboratory investigations that help in their diagnosis are urine
testing for the presence of increased amount of GAGS and assay
of suspected enzymes in white cells, fibroblasts or sometimes in
serum. In certain cases a tissue biopsy is performed and the
GAG that has accumulated can be determined by electrophoresis
(Neufeld and Muenzer, 1995).
Hyaluronidase is one important enzyme involved in the
catabolism of certain GAGS that has been implicated in any
mucopolysaccharidosis . Because of the extended nature of the
polysaccharide chains of GAGS and their ability to gel, the
proteoglycans can act as sieves restricting the passage of large
macromolecules into the extracellular matrix but allowing
relatively free diffusion of small molecules (Hardingham TE,
and Fosang 1992).
56

Review of Literature
Vernier and brown, 1983 stated that the conentraions of
anions in the GBM decrease in patients with congenital
nephritic syndrome and they proposed that the basic defect in
the syndrome is the failure to incorporate HS into the
macromolecular structure of the GBM resulting in loss of HS
into the urine.
It was found that the major determinants of the charge
dependent permeability of the glomerular filter are
glycosaminoglycans- (GAG) (Kery, 1992).
In normal subjects GAG can be detected in considerable
amounts in urine but only in very low concentration in the
serum, the main components in normal urine are chondriotin
sulfate and heparan sulfate (HS), while dermatan sulfate (DS) is
found only in small amounts. Urinary GAG represent a
heterogenous mixture of partially depolymerized and partially
desulfated products of tissue glycosaminoglycans (DeMuro, et
at., 2001).
Evaluation of renal biopsy in diabetic nephropathy showed
a reduction in HSPG content, thus confirming the role played by
PG in the control of glemerular permeability due to their
negative charge (Vernier, et al., 1992).
57

Review of Literature
The GAG excretion per functioning glomerular area
calculated as functional GAG excretion (FGE) was decreased in
all the glomerulonephritides compared to both healthy controls
and diabetic nephropathy (Tencer et al., 1997).
Elevated urine GAG has been suggested as a marker of
glomerulonephritis (Bagio and cicerello, 1988).
A role for HSPG has been demonstrated for binding of
DNA containing complexes to the glomerular basement
membrane (GBM) (Kramers, et al., 1994).
Heparan sulfate could be one of autoantigens in SLE
because it serves as a target antigen for in vivo cross reactive
anti- DNA antibodies (Suzuki, etaL, 1993).
It has been suggested that HS might serve as a target
antigen invivo for cross ~ reative anti DNA antibodies. It has
also postulated that auto immunity to HS may be responsible for
the induction of tissue damage and kidney dysfunction in SLE
(Kashihara, et al., 1992).
It was found that, the reactivity of anti- HS antibodies or a
cross- reactive anti DNA-antibodies with HS may induce
glomerulonephritis in mice by triggering inflammatory reactions
locally. Alternatively, direct binding of anti - HS or anti DNA
antibody to HS expressed on the endothelial cells on the GBM
may stimulate release of several inflammatory cytokines by EC
and mesangial cells (Maruyama, et al., 1993).
58

Review of Literature
Alteration of the distribution pattern and composition of
glycosaminoglycans (GAG) and' proteoglycans may play an
important role in the development of autoiumune disease.
Recent experiments indicate that anti - DNA antibodies cross
reacting with hyaluronic acid, heparan sulfate and chondriotin
sulfate are present in patients with systemic lupus
erythematosus, (Hansen, et al., 1996).
The prescence of anti – heparan - sulfate (HS) reactivity in
serum is closely related to the occarance of nephritis in patients
with systemic lupus erythematosus (Kylkema, et al., 1995).
It was found that experimental systemic lupus
erythematosus (SLE) -like disease was induced in BAIB/C mice
by immunization with heparan sulfate, the major
glycosaminoglycans of glomerular basement membranes (Ofosu
Appiah, et al., 1998).
Recently, a grin was identified as a major HSPG present in
the glomerular basement membrane (GBM) An increased
permeability of the GBM for proteins after digestion of HS by
heparitdinase or after antibody binding to HS demonstrated the
importance of HS for the permselective properties of GBM. It
was demonstrated that there is a decrease in HS staining in the
GBM in different human proteinuric glomerulopathies such as,
SLE, Minimal change disease. Membranous glomerulonephritis,
and diabetic nephropathy (Raats, et al., 2002).
59

Subjects and Methods
Patients:
Subjects and Methods
Fourty subjects were examined at the outpatient and
inpatient clinics of Rheumatology and Rehabilitation
departments, Zagazig University Hospitals, they were divided
into the following groups;
Group I:
It included 30 patients suffering from SLE, they were 29
females and one male, their ages ranged, form (16 to 45 ys) with
a mean value of (26.2 ± 8.2) years. Disease duration ranged
from (6 months to 11 years) with a mean value of (3.3 ± 2.7)
years.
They were diagnosed according to the American
Rheumatism Association (ARA) revised criteria for
classification, of SLE (Tan, et al., 1982) Which include:
1-Malar rash.
2- Photosensitivity.
3- Discoid rash.
4- Oral ulcers.
5-Arthritis.
6- Serositis:
a- Pleuritis or b- Pericarditis.
60

Subjects and Methods
7- Renal disorder:
a- Persistent proteinuria greater than 0.5 gm per day or
greater than 3+ if quantitation not performed or
b-Cellular casts, may be red cells, heamoglobin,
granular, tubular or mixed.
8- Neurologic disorder:
9- Heamatologic disorders:
a- Seizures or b- Psychosis.
a- Heamolytic anemia with reticulocytosis or
b- Leucopenia less than 4000/mm 3 total on two or more
occasions or
c-Lymphopenia less than 1500/mm 3 on two or more
occasions or,
d- Thrombocytopenia less than 100.000/mm 3 in the
absence of offending drugs.
10- Immunologic disorders:
a- Positive LE cells or
b- Anti-DNA antibody to native DNA in abnormal titre or.
c- Anti Sm: presence of antibody to Sm nuclear antigen or
d- False positive serologic tests for syphilis.
11- Antinuclear antibody: an abnormal titre of antinuclear
antibody by immunofluorescence.
61

Subjects and Methods
Any patient had any four or more of eleven criteria is said
to have the disease.
Patients were under treatment with corticosteroids, imuran
and some of them received pulsed I.V cyclophosphamide.
18 patients had lupus nephritis, diagnosed on the basis of
proteinuria, haematuria & Laboratory investigations, (increased
anti DNA & impaired renal functions).
Selection of patients:
All patients didn't suffer from any other illness that might
affect urinary GAG as: e.g. Diabetes mellitus, liver
disease, Mucopolysaccharoidosis.
Group II:
Ten healthy subjects were taken as a control group. They
were 2 males and 8 females, their ages ranged from (20-45)
years with a mean value of (29,0 ± 9.7).
All patients were subjected to an examination sheet with
stress on the following:
62

Subjects and Methods
A -Full history taking which include:
- Photosensitivity.
- Arthritis or arthralgia.
-Falling of hair.
- Fever, fatigue, malaise.
- Oral ulceration.
- Vasculitic changes.
- Loin pain.
-Nodules.
- Dysuria or haematuria.
- Seizures, headache, or phychosis.
- Past history of abortion or still birth.
B- Complete clinical examination for every subject was
done including the following:
1-General examination including pulse ,blood pressure,
temperature,……….
2- Local examination including joint for warmth,
tenderness, swelling and deformity ,crepitus……...
3- Skin examination including skin rash.
4- Cardiovascular examination. 5- Chest examination.
63

Subjects and Methods
6- Examination for manifestations of renal involvement as
hypertension and oedema of L.L.
7- Neurological examination.
8- Abdominal examination for lymphadenopathy.
C- Investigations: included:
1- Complete blood picture including differential cell count.
2- Erythrocytic sedimentation rate (Dacie and Lewis, 1995).
3- Complete urine analysis
4- Kidney function tests:
•Urea: was measured using kinetic UV assay:(Teitz,1995)
•Creatinine: was measured using kinetic, colorimetric
assay:(Foster-Swanson et al, 1994).
5-24 hour proteinuria.
6-ANA.
7- Anti DNA antibody: done by indirect fluorescent test for
detection of anti-nDNA in human sera. (Virgo reagents
supplied by electronucleonics, Incs, Washington) (positive
value up to 25 Lu/ml).
8- Liver function tests.
9- Fasting blood sugar.
10- Radiology: plain X-ray pelvis and chest for evidence of
renal stones, pleural or pericardial effusion.
64

Subjects and Methods
Method for estimation of urinary glycosaminoglycans
(GAGs)
GAG were isolated from urine by ion exchange
chromatography on DEAD- Sephacel (Pharmacia) according to
Staprans et al. (1981). Fifty ml of urine were centrifuged at
5.000 g for 15 min and applied directly to a DEAD Sephacel
column (0.7 x 8 cm) equilibrated with 0.15 M NaCl buffered
with 0.02 M Tris-HCl, pH 8.6. After extensive washing of the
column with the equilibrating buffer, the adsorbed material was
eluted with 2 M LiCl and 0.02 M Tris-HCl, pH 8.6. Ten
fractions (1 ml each) were collected and analyzed for their
uronic acid content . All of the hexuronate-containing fractions
were pooled and GAG were precipitated with 4 volumes of
ethanol at 4°C, The mixture was left overnight and the
precipitated was separated by centrifugation at 8.000 g for 15
min, washed twice with ethanol and dried.
The GAG composition was determined, after
solubilization with water, by electrophoresis on acetate cellulose
strips in a discommons buffer Bitf-composition was expressed
in terms of relative percentages based on densitometric scanning
of Alcian Blue stained strips using a Scan Analysis Program
(Thunder Scan-Biosoft). GAG identification was performed by
treating aliquots of the samples (containing about 100 μg of
65

Subjects and Methods
hexuronate) at 37°C for 18 hrs before electrophoresis with
specific eliminases. All enzymes were purchased from Sigma.
The specificity and efficiency of the enzyme treatment
were checked using a standard GAG (Sigma) under the same
experimental conditions. In some cases, aliquots of samples
were freed of protein by papain treatment (150 μg/mg protein)
in 0.1 M sodium acetate buffer, pH 6.2 containing 5.0 mM
cysteine and 5.0 mM EDTA at 56°C for 48 hrs, precipitated
with ethanol. Solubilized with water and submitted to
electrophoresis. (Staprans et al., 1981)
Enzyme-linked immunosorbent assay of heparan
sulfate and chondriotin sulfate
Step 1: Each well in the Falcon 96 well plate was precoated to
100 μl of 0.5 mg/ml protamin chloride per well and over
night at 4 o C. After washing with PBS, each was added
100 μl of HS and CS solutions per well (50 mg of HS and
50 mg of CS in 1000 ml of PBS, containing 0.5% BSA)
followed by incubation over night at 4 o C.
Step 2: HepSS-1 was diluted two hundred fold with PBS and the
urine sample was diluted three fold with PBS. Solutions
containing serially decreasing concentration of HS and
CS standards or samples were mixed with the same
66

Subjects and Methods
aliquot (0.2 ml) of HepSS-1 solution and incubated for 2
hours at room temperature.
Step 3: After washing the plate from step 1 with PBS
containing 0.05% Tween 20, the solution from step 2 (0.1
ml) was added to each well and inclubated for 1 hour at
room temperature. After washing with PBS, the bound
antibody was detected by the immunoperoxidase
procedure using a Vectastain ABC kit for mouse IgM. O-
phenylenediamine dihydrochloride was used for the
superature, the signal was read spectrophotometrically at
492 nm by micro plate reader. (Staprans et al., 1981)
67

Subjects and Methods
Disease activity in SLE patients
It was assessed using disease activity parameters of a
graded disease activity index called: University College
Hospital/Middle sex criteria (UCH) described by “Morrow et
al., 1982” which is illustrated in table ( IX)
Table ( IX ): The UCH/Middle sex score.
Horizontal 10 cm visual analogue scale of wellbeing
(5 cm or an increase > 2 cm since previous visit).
Pyrexia (> 37.5) not due to infection
Lymphadenopathy not due to infection
Arthralgia and /or Myalgia
Pleuritis and/or pericarditis
Vasculitis skin rash grade 1
2
3
Raynaud’s phenomenon
Cerebral involvement grade 1
2
3
Renal proteinuria (+ or more)
proteinuria trace + hypertension
proteinuria trace + stable urea/creatinine
increase proteinuria ± rising BP ± rising creatinine
Easy bruising/bleeding
Steroids:
Prednisolone: 0- 4 mg/day
5- 24 mg/day
25 mg+/day
68
Score
1
1
1
1
2
0
1
2
1
1
2
3
1
2
3
1
0
1
2

Subjects and Methods
Grading of skin involvement:
Grade 1: < 4/9 body surface involved, no infraction or grade 2
or 3 in healing phase.
Grade 2: > 4/9 body surface involved, no infarction or
ulceration or trophic changes.
Grade 3: Any distribution with infarction, ulceration or trophic
changes.
Grading of cerebral involvement:
Grade 1: Frequent vascular headaches and / or visual
phenomenon or grade 2 or 3 recovering.
Grade 2: Disturbance of Mood or clouding of consciousness
with normal functioning.
Grade 3: Neurological deficit developed within last month or
disturbance of mood or clouding of consciousness
inconsistent with normal function.
Scoring Total score
Grade I In
active
69
0-1
Grade II Mild 2-4
Grade III Moderate 5-7
Grade IV Severe 8+

Subjects and Methods
70

Subjects and Methods
We divided our patients according to disease activity index
into 2 groups: table (2)
Group 1: inactive 56.7%.
Group II: Active: a- Mild active 23.3%
b- Moderate active 10%.
c-Severe active 10%.
We redivided them according to renal involvement into 4
subgroups as follow:
(I) Inactive: a- With renal involvement (23.3%).
(II) Active;
b- Without renal involvement (33.3%).
a -With renal involvement (20/%)
b-With out renal involvement (23.3%)Table (3)
71

Subjects and Methods
Statistical Analysis
All data were coded. Entered and analyzed using EPI-
INFO (version 6.1) software computer package (Dean et al.,
1994).
I-Arithmetic mean [X]:
Where:
X
X
n
X = sum of individual data.
n = number of individual data.
II- Standard deviation [SD]:
Where:
SD
X 2
n 1
X n
2
2
X = sum of the squares of individual values.
(X) 2 = the square of the sum of values.
III-Student [t] test:
Used to test for the significance between two means
according to the following formula.
t
2 SD SD
n
1
1
X
1
X
2
n
2
2
2
72

Subjects and Methods
Where:
X1, X2 = the mean of first and second groups respectively.
n1, n2 = the number of first and second groups
respectively.
SD1, SD2 = the standard deviation of the first and second
groups respectively.
The results of the (t) value was then checked on, using
student’s (t) table to find out the levels of significance, the
probability for a (t) value (P value)
P > 0.05 non-significant
P < 0.05 significant
P < 0.01 highly significant
IV-Chi-Square [X 2 ] test:
Used to test for the significance between proportion
according to the following formula:
Where:
X
2
O_E E
= summation
O = Observed value
Column total
row total
E
overall total
2
73

Subjects and Methods
The probability (P) is then obtained from the (X 2 )
distribution tables according to a certain degree of freedom
(D.F) = (number of columns-1) (number of rows-1).
N.B.: For 2 x 2 tables when the expected cell is less than 5,the
Chi square Yates correction (X 2 , y) was used.
V-Correlation: measures the closeness of the association.
r =
X XY
Y
XX YY
Where X/Y denotes the values and X/Y are their
corresponding means.
The correlation coefficient (r) is +ve when X/Y tend to be
high or low together, (r) is -ve when high values of X go with
low value of Y.
VI- Analysis of Variance: (ANOVA or F test) for
comparison of means of more than two groups. F-value
was calculated according to the following formula:
Source of
Variation
Sums of squares (SS)
Degrees of
freedom
Mean square MS F ratio
A
N
2
K-1
ss A MsA =
K 1
MS A F =
MSW
Within groups SSW =SST –SSA N-K
SSW MSW =
N K
2
Among groups X
ss n X
Total SST=
X
X 2
2 N-1
N
74

Subjects and Methods
Where:
N = total number of observations in all groups.
N = number of observation in each group.
K = number of groups.
The significance level (P-value) of "F" was obtained from "F"
tables.
If the F value is significant, least significant difference (LSD) is
calculated at different probability values as follows:
L.S.D. 0.05 = t 0.05 / n 1/ n .......
MS W
0.01 0.01
0.01 0.001
1 1 2
75

Discussion
Discussion
Systemic lupus erythematosus (SLE) is a complex auto
immune disease that can involve multiple organ system. The
kidney is the most common visceral organ affected by SLE
(Trouw, et al 2004).
Approximately, one third of membranous
glomerulonephritis (MGN) cases in adults are associated with
systemic disease including systemic lupus erythematosus (SLE).
(Lin, et al 2003).Lupus nephritis remains a major cause of
morbidity and mortality in patients with systemic lupus
erythematosus "Cortes -Hernandez,etal,2003"
Jacobsen, et al 1998 stated that progression of
nephropathy in their SLE patients to chronic renal insufficiency
or end stage renal disease occurs in 45% and 12% respectively.
Glycosaminoglycans (GAGs) are heteropolysaccharides
present as integral components of the extracellular matrix
(ECM), cell and basement membranes. GAGs play an important
role in immune and inflammatory responses because of their
ability to interact with cytokines and chemokines, promoting the
localization of these molecules onto the ECM or cell
membranes at specific anatomical sites (Fernandez, et al 2002).
93

Discussion
Since GAGs orginate from different kinds of connective
tissues, their measurement in urine may be useful to evaluate the
metabolic state of various organs Ilhan, et al 2003.
Heparan sulfate protaglycan is an important component of
the glomerular anionic filtration barrier Edge and spiro, 2000.
The urine contains several forms of GAGs but the vast
majority of them are either chondriotin sulphate or heparan
sulphate Tencer, et al,.1997.
Our work was to evaluate glycosaminoglycans (GAG).
level in urine of systemic lupus erythematosus patients with and
without renal involvement and its role as a marker for lupus
nephritis with its correlation to disease activity.
In our work, when studying clinical characteristics of
diseases activity in our SLE patients we found that Raynaud’s
phenomenon present in 33.3% of our patients.
This coincided with Michael Belmont, 1998 who stated
that Raynaud’s phenomenon is observed in about 30% of
patients with SLE.
We also found that arthralgia and/or myalgia present in
only 16.7% of our patients.
94

Discussion
These results disagreed with the study done by Emery
2004 who stated that arthralgia and/or myalgia present in
approximately 95% of the SLE patients.
This contrast may be found due to the few number of the
patients included in our study and most of them were in disease
remission (inactive patients were seventeen).
In this work, we found that anti-DNA antibody was
positive in about 43.3% of our patients.
This coincided with Micheal Belmont 1998, who stated
that 30 to 70% of his SLE patients were anti DNA positive.
In this study, we found that the total GAGs levels, HS and
CS levels were higher in severe active subgroup of SLE
patients.
In contrast Parildar, et al., 2003 found that there were no
correlations between GAGs and SLE. DAI scores.
This contrast may be due to the use of different disease
activity index.
In our work, there was highly significant difference (P <
0,001) between total urinary GAG level and the presence of
arthritis.
95

Discussion
This disagreed with Ilhan, et al 2003, who found that
urinary GAG levels are unrelated to the presence of arthritis.
Our results showed non significant correlation (P> 0.05)
between urinary GAG level and other laboratory parameters
including ESR, Anti DNA, 24 hour proteinuria and creatinine
level.
This coincided with the results of the study done by De
Muro, et al 2001 who found that there is no correlation (P>0.05)
between the total urinary GAGs concentration and the ESR , 24
hour proteinuria and Anti DNA.
In contrast, Ilhan et al, 2003 found a significant
correlation between the total urinary GAGs and Anti DNA titre
in lupus nephritis patients.
In our work, it was found that there was significant
difference (P

Discussion
This also coincided with the results of the study done by
Ilhan, et al, 2003 and De Muro, et al 2001 who found that
urinary GAGs levels were higher in lupus patients than in
control group.
In our work, we found that total urinary GAGs levels were
higher in active patients with extra renal disease compared to
other subgroups of SLE patients and control group.
These results are in agreement with De Muro- et al , 2001
who found that urinary GAGs levels were higher in patients
with extra renal disease compared to control group and they
hypothesized that this could have been due to an activation of T
lymphocytes.
In contrast, Parildar, et al, 2002 found that total urinary
GAG levels were higher in Lupus nephritis patients and they
found that GAGs values in class 3 nephritis were significantly
(P < 0.05) higher than both class 2 and class 4 lupus nephritis.
This contrast may be due to the use of high dose of
immuno-suppressive therapy in our patients with renal
manifestations which may cause a decrease in GAG synthesis
by T lymphocytes.
These results also disagreed with the results of the study
done by Ilhan, et al, 2003 they have observed increased total
urinary GAGs levels in lupus nephritis patients.
97

Discussion
Van den Born et al, 1992 found increased GAGs
distribution in areas with mesangial matrix production and this
suggests the production of GAGs by mesangial cells in
glomerular diseases thus, it is supposed that in diseases with
enhanced mesangial proliferation, such as mesangial
proliferative glomerulonephritis or IgA nephropathy, the
synthesis of GAG is up regulated.
In our study, there was significant difference (P

Discussion
Ilhan et al, 2003, found in their study that, HS levels in
urine were significantly higher in lupus nephritis.
Also in this work, we found that the ratio between CS/ HS
is higher in active SLE patients than inactive patients.
These results agreed with the results of the study done by
De. Muro et al 2001 who found that CS /HS ratio was
significantly reduced in patients with SLE remission. And they
found a difficulty in explaining this result and they hypothesized
that it might indicate tubular or interstitial rather than a
glomerular lesion. Lupic et al., 1986, propsed the use of the CS/
HS ratio as a semiquantitative index of tubular damage
alternative to urinary protein electrophoresis on polyacryl amide
gel.
We found in our study that CS/ HS ratio was higher in
Active patients with renal involvement.
But these results are in disagreement with the results of the
study done by De Muro, et al 2001 who observed that CS /HS
ratio was independent of renal disease.
99

Summary & Conclusion
Summary:
Summary and Conclusion
Systemic lupus erythematosus (SLE) is a multisystem
autoimmune disorder characterized by multiorgan pathology and
autoantibodies against a variety of autoantigens.
Lupus nephritis remains a major cause of morbidity and
mortality in patients with systemic lupus erythematosus.
Glycosaminoglycans (GAGs) are major components of the
extracellular matrix and they have key roles in fibrotic diseases.
This work was done in Rheumatology and Rehabilitation,
and Biochemistry departments of Zagazig University Hospitals
to evaluate the relation of the total urinary level of
glycosaminoglycans, heparan sulfate and chondriotin sulfate
levels to the disease activity and their relation to renal affection
in SLE patients.
This study comprised a total of 40 subjects, they were
divided into 2 groups:
1- Group I: included 30 patients suffering from SLE.
2- Group II: included 10 normal volunteers.
100

Summary & Conclusion
Our patients were collected according to the American
Rheumatism Association (ARA) revised criteria for
classification of SLE (Tan, et al., 1982) and their age ranged
from (16-45) ys.
All patients were subjected to full history taking with
stress on arthritis or arthralgia, fever, vasculitic changes, Loin
pain, dysuria, seizures, headache or psychosis.
We divided our SLE patients according to disease activity
index into 2 groups: table (2)
Group I: inactive 56.7%.
Group II: Active 43.3% : a- Mild active 23.3%
101
b- Moderate active 10%.
c-Severe active 10%.
We redivided them according to renal involvement into 4
subgroups as follow:
(I) Inactive: a- With renal involvement (23.3%).
(II) Active;
b- Without renal involvement (33.3%).
a -With renal involvement (20/%)
b-With out renal involvement (23.3%)Table (3)

Summary & Conclusion
All patients were clinically examined with stress on:
General examination, joint examination, skin rash,
cardiovascular and chest examination, oedema of L.L.,
lymphadenopathy.
All cases were subjected to laboratory investigations including:
1- Complete blood picture.
2- Erythrocytic sedementation rate.
3- Complete urine analysis.
4- Kidney function tests.
5- 24 hour proteinuria.
6- ANA.
7- Anti DNA antibody.
8- Liver function tests.
9- Fasting blood sugar.
10- Plain x ray pelvis & chest.
11- Urinary glycosaminoglycans levels (ion exchange
chromatography on DEAE- Sephacel) including HS &
CS levels. by (Enzyme-linked immunosorbent assay)
102

Summary & Conclusion
The following results were obtained:
- Among clinical cnaracteristicas of disease activity in our
SLE patients Raynaud’s phenomenon was the most
common feature (33.3%) while pyrexia and serositis both
affected only 3.3% of patients.
- ESR level was higher in sever active patients anti
DNA titres were higher in active SLE patients than
inactive ones .
- CS and Hs levels were higher in severe active
patients
- Ahighly significant difference between positive and
negative cases of arthralgia, vasculitic skin rash and
lymphadenopathy as regard total urinary GAG level. There
was significant difference between positive and negative
cases of Raynaud's phenomenon and neuropsychiatric
manifestations as regard total urinary GAG level while
there was no statistical difference between positive and
negative cases for pyrexia, seroitis and bleeding tendency
as regard total urinary GAG level.
- Non significant correlation between urinary
GAG level and other laboratory parameter:
including ESR, Anti DNA, 24 hour proteinuria
and creatinine level.
103

Summary & Conclusion
- A significant elevation of total urinary GAG in SLE
patients compared to control group.
- A significant elevation of urinary HS in SLE patients
compared to control group while there is no statistical
difference between the two groups as regard CS level.
- A highly significant difference between SLE patients and
control group as regard CS/HS ratio.
- A significant elevation of total urinary GAG in active SLE
patients with extrarenal disease compared to inactive
patients.
- A significant elevation of HS and CS levels in active SLE
patients compared to inactive patients.
- A significant difference between active SLE and inactive
patients as regard CS/HS ratio.
104

Summary & Conclusion
Conclusion
In conclusion, we believe that analysis of urinary GAG
may represent an additional, non invasive diagnostic approach
in SLE patients because it might indicate the presence of an
early abnormal permeability of the renal filter in patients
without other appreciable signs of kidney alteration.
105

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129

Appendix
(1) Presonal history:
Sheet
Name Address
Sex Occupation
Age Habit
Marital state
(2) C/O:
(3) Present history:
130
Croup :
Case No:
Date:
Onset : Course : Duration:
Fatigue
Fever : Onset
- Weight loss
- M.S:- é ttt
- é out ttt
Course
Duration
Ch.ch
Associated symptoms
Photosensitivity: Malar rash :
Falling of hair: Discoid rash :
Skin rash :
Vasculitis : - Trophic changes: - Purpura:
- Ulcers : - Nail fold hges :

Appendix
Nodules : - Raynaud’s phenomenon:
- Oral Ulcers:
Eye manifestations:
Joint affestion :
- Dryness:
-Redness :
Medications :
Other systems :
Types:
Course :
Doses :
Duration :
Interactions:
1- Kidney : -Dysuria
-Haematuria
-Loin pain
2- Chest : - Cough - Expectoration
3- Heart :
- Chest pain
- Palpitation - Dyspnea
4- Abdominal : - Dry mouth - Epigastric pain
- Dysphagia - Nausea & vomiting
- Heamatemesis & Melena
5- CNS : - Headache
- Depression
- Psychosis
- Neuropathy
- Sizeures
3- Menstrual history:
-Time of 1 st menarche -Amenorrhea
4- Past history - Pregnancy - Drugs
- Abortion
- Still birth
- Contraceptive pills
5- Family history: - Similar condition
131

Appendix
Examination
1- General exam
- Pulse - Body weight
- Bl. Pr - Body temprature
- General appearance
- Hepatosplenoermegaly
2- Systemic examination :
1- Cardiac
- Pericardical rub
2- Pulmonary (chest)
- Pleural rub
- Crepitations
3- Neurological examination
- Cranial nerve affection
- Mononeuritis multiplex
- Tone - Power - Reflexes
4- Locomotor systeme
A- Joint examination:
Inspection: Deformity
Swelling
Wasting
Skin over :sinus, redness
Palpation: Tenderness
Hotness
Swelling: Effusion
Synovial thickning
ROM
B- Muscle examination :
Myalgia
5- Skin examination
Alopecia
Nails
Malar rash
Mucous membrane ulcers
Non specific rash
132

Appendix
1- Plain x ray :
Investigations
2- Blood picture & Differential cell count space
3- ESR & CRP
4- complete urine analysis
Albumin protein in 24 hour urine.
Abnormal casts aspect
Pus cells colour
5- Serum urea & creatinine
6- ANA
7- Antids DNA antibodies
8- Liver function tests
9- Fasting blood sugar
10- GAG in urine
11- HS level
12- CS level
13- Ratio : HS/ CS
133

Appendix
The UCH /Middlesex score
Horizontal 10 cm visual analogue scale of well-being (5 cm or
an increase > 2 cm since previous visit).
134
S1core
Pyrexia (> 37.5 °C) not due to infection 1
Lymphadenopathy not due to infection 1
Arthralgia and/or myalgia. 2
Vasculitis skin rash grade 1 0
2 1
3 2
Raynaud’s phenomenon. 2
Cerebral involvement grade 1 1
Renal proteinuria (+ or more)
2 2
3 3
Proteinuria trace + hypertension. 1
Proteinuria trace + stable urea/creatinine. 2
Increase proteinuria ± Rising BP ± Rising creatinine 3
Easy bruising / bleeding 1
Steroids:
Prednisolone 0- 4 mg/day 0
Scoring:
5-24 mg/day 1
25 mg + / day 2
Grade I Inactive
Total score
0-1
Grade II Mild 2-4
Grade III Moderate 5-7
Grade IV severe 8+
1

Recommendation
We recommend to evaluate the urinary GAG
excretion in a group of untreated SLE patients and
studying a larger number of patients with close
observation and evaluation.

Arabic Summary
يبرعلا صخلملا
ةزھجأ نم ديدعلا يلع رثؤي نمزم يعانم ضرم وھ ءارمحلا ةبئذلا ضرم
١
-:
ةمدقملا
رمدت باھتللاا طئاسو عم اھرودب يتلاو ةيتاذ ةداضم ماسجأ جاتنإب زيمتيو مسجلا
يف ةافولل يساسأ ببس ربتعي يلكلا يلع ءارمحلا ةبئذلا ضرم ريثأت نأو
. ةجسنلأا
. ضرملا اذھب نيرثأتملا ىضرملا
يجراخلا بلاقلا نم يسيئر ءزج ربتعي زناكيلجونيمأ زوكيلجلا بكرم نأ
. ةيفيللا ضارملأا يف ماھ رود بعلي وھو ايلاخلل
: ثحبلا نم فدھلا
لوبلا يف زناكيلج ونيمأ زوكيلجلا تابكرم ةبسن سايق وھ لمعلا اذھ فدھ
ضرملاب يلكلا رثأتب ًاضيأ هتقلاع مييقت و ءارمحلا ةبئذلا ضرم طاشن يدمب هتقلاعو
-:
ثحبلا قرطو داوم
ةرشعو ءارمحلا ةبئذلا ضرم نم نوناعي ًاضيرم ٣٠ يلع ثحبلا اذھ يرجأ
. ةللحملا داوملا ةيحلاص مييقتو ةنراقملل نيرخآ صاخشأ
مزيتامورلا مسقل يلخادلا مسقلاو ةيجراخلا ةدايعلا نم ىضرملا عمج مت دقو
ًاماع
٤٥-١٦
نيب ام مھرامعأ تحوارت دقو قيزاقزلا
ةعماج تايفشتسمب ليھأتلاو
. ١٩٨٢
-:
ماعل
Tan
ملاعلا صاوخل ًاعبت مھعمج مت دقو
ةيتلآا تاعومجملا يلإ ىضرملا ميسقتب انمق دقو
.
طيشن ريغ -:
-:
ضرملا طاشن بسح -١
يلولأا ةعومجملا
-

Arabic Summary
٢
. طيشن -:
ةيناثلا ةعومجملا
تاعومجم ٤ -:
ضرملاب يلكلا رثأت بسح -٢
. يلكلا رثأت عم طيشن ريغ -١
. يلكلا رثأت مدع عم طيشن ريغ -٢
. يلكلا رثأت عم طيشن -٣
. يلكلا رثأت مدع عم طيشن -٤
-:
-
يتلأا لمع مت دقو
باھتلا وأ لصافملاب ملاآ دوجوب مامتھلاا عم لماك ضرم خيرات ذخأ -١
. عارص وأ تاجنشت ، نطبلاب ملأ ، ةرارحلا ةجرد عافترا لصافملاب
لصافملا صحف و ماعلا صحفلا
يسفنتلاو يرودلا زاھجلل صحف،
-:
يلع زيكرتلا
عم لماك يكينيلكإ صحف -٢
نيمدقلاب مروت دوجو
،
يدلج حفط دوجو
. ةيوافميللا ددغلا يف مخضت دوجو
-:
ةيتلآا ةيلمعملا ثوحبلا -٣
. ةلماك مد ةروص
. ءارمحلا مدلا تارك بيسرت لدعم
. ةعاس
. يلكو دبك فئاظو
٢٤ لوب يف نيتوربلا يوتسم
مدلا يف ركسلا
يوتسم
هيإ نإ هيإ ـلا ضمحل ةداضملا ماسجلأا
.
هيإ نإ يد ـلا ضمحل ةداضملا ماسجلأا
-
-
-
-
-
-
-

Arabic Summary
نارابيھلا نم لكو
،
زناكيلج ونيمأ زوكيلجلا بكرم يوتسم سايق
. لوبلا يف تيفلس نيتويردنوكلاو تيفلس
٣
-:
-
ثحبلا اذھ جئاتن
نمزناكيلج
ونيمأ زوكيلجلا بكرم يوتسم نيب ةبلاس ةيئاصحإ ةقلاع دوجو -١
ةداضملا ماسجلأاو ءارمحلا مدلا تارك بيسرت لدعم نم لك نيبو لوبلا
لدعمو ةعاس
٢٤
لوب يف نيتوربلا يوتسمو هيإ نإ يد ـلا ضمحل
. نينتيايركلا
لوبلا يف زناكيلجونيمأ زوكيلجلا بكرم يوتسمل ةميقلا وذ عافترا ادجو -٢
. ءاحصلأا صاخشلأاب ةنراقملاب ءارمحلا ةبئذلا ضرمل
ةبئذلا ضرمل لوبلا يف تيفلس نارابيھلا يوتسمل ةميق وذ عافترا
دجو -٣
يوتسمل ةميق وذ عافترا دجوي مل امنيب ءاحصلأا صاخشلأا ةنراقملاب ءارمحلا
صاخشلأاب ةنراقملاب ءارمحلا ةبئذلا ضرمل لوبلا يف تيفلس نيتويردنوكلا
. ءاحصلأا
نيتويردنوكلا و اتيفلس
نارابيھلا بكرم نيب ةبسنلل ةميقلا يلاع عافترا دجو -٤
. ءاحصلأا صاخشلأاب ةنراقملاب ءارمحلا ةبئذلا ضرم يف ايفلس
لوبلا يف زناكيلج ونيمأ زوكيلجلا بكرم يوتسمل ةميق وذ عافترا دجو -٥
ءانثأ نيرخآ ضرمل ةنراقملاب ضرملا طاشن ءانثأ ءارمحلا ةبئذلا ضرمل
تيفلس نارابيھلا بكرم
. ضرملا سفن نوكس
يوتسم نم لكل ةميق وذ عافترا دجو امك -٦
ءانثأ ءارمحلا ةبئذلا ضرمل امھنيب ةبسنلاو لوبلا يف تيفلس نتيويردنوكلاو
.
ضرملا سفن نوكس ءانثأ نيرخآ ضرمب ةنراقملاب ضرملا طاشن

Arabic Summary
لوبلا يف زناكيلجونيمأ زوكيلجلا بكرم يوتسمل ةميق وذ عافترا دجو -٧
ةنراقملاب ضرملاب يلكلا
يف رثأت نم نوناعي لا نيذلا ءارمحلا ةبئذلا ىضرمل
. ضرملا سفنب يلكلا رثأت نم نوناعي نيرخآ ىضرمب
ةبئذلا يضرمل لوبلا يف زناكيلجونيمأ زوكيلجلا بكرم ىوتسم سايق
٤
-:
نأ جتنتسأ دقو
ةبسنلاو لوبلا يف تيفلس نيتويردنوكلاو تيفلس نارابيھلا نم لك سايقو ءارمحلا
سفنب يلكلا رثأتو ضرملا طاشنل
ليلدك همادختسا نكمي يضرملا سفنل امھنيب
.
ةعانملل ةطبثملا ةيودلأاب جلاعلا تحت نوكي لاأ طرشب ضرملا

ىف لوبلا ىف زناكيلجونيمأ زوكيلجلا بكرم
ىلكلا رثأتل رشؤمك ءارمحلا ةبئذلا ىضرم
ضرملا طاشن ىدمب هتقلاعو
قـــيزاقزلا ةـــعماج -بــــطلا
ةيلك ىلإ ةمدقم ةـــــلاسر
ليھأتلاو مزيتامورلا بط ىف ريتسجاملا ةجرد ىلع لوصحلل ةئطوت
دامح سابع ىحتف حامس
روتكدلا ذاتسلأا
دجملا وبأ ىرسي
ةيويحلا ءايميكلا مسقب ذاـــتسأ
قيزاقزلا ةعماج – بـطلا ةــيلك
روتكدلا
ةريمع معنملا دبع دمحأ
ليھأتلاو مزيتامورلا سردم
قيزاقزلا ةعماج – بـطلا ةــيلك
ةــــحارجلاو بـــطلا سوـــيرولاكب
قــــيزاقزلا ةـعماج – بـــطلا ةيلك
نوفرشملا
بـــــــطلا ةــــــــيلك
قـــيزاقزلا ةـــعماج
٢٠٠٤
/ ةبيبطلا نم
ةروتكدلا ةذاتسلأا
ميلس باھولا دبع ةبھ
ليھأتلاو مزيتامورلا دعاسم ذاـــتسأ
قيزاقزلا ةعماج – بـطلا ةــيلك
ةروتكدلا ةذاتسلأا
هتاحش ايركز ةميمأ
ليھأتلاو مزيتامورلا دعاسم ذاـــتسأ
قيزاقزلا ةعماج – بـطلا ةــيلك