The Islamic University – Gaza Deanship of Graduate Studies Faculty of Science Biological Science Master ProgramMedical technology

Comparison of Anti Cyclic Citrullinated Protein 2 Serum Levels with Rheumatoid Factor for the Diagnosis of

Rheumatoid Arthritis in Gaza Strip

Prepared by

Bushra M. Skaik

Supervisor

Prof. Mohammad E. Shubair

Submitted in Partial Fulfillment of Requirement for the degree of Master of Life Sciences/Medical Technology

1429ه - 2008م

Declaration

“I herby declare that this submission is my own work and that, to the best of my

knowledge and belief, it contains no material previously published or written by

another nor material which to a substantial extent has been accepted for the

award of any other higher degree or diploma of the university or other institute of

higher learning, except where due acknowledgement is made in the text"

Author

Bushra Mohammed Skaik

Signature: bushra Date: Feb- 2008

.

All Rights Reserved © 2008. No part of this work can be copied, translated or

stored in retrieval system, without prior permission of the author.

I

Comparison of Anti Cyclic Citrullinated Protein 2 Serum Levels with Rheumatoid Factor for the Diagnosis of Rheumatoid

Arthritis in Gaza Strip

Abstract

II

Background: Rheumatoid Arthritis (RA) is the most common autoimmune

disease. Howevere, specific and practicable tests for its specific diagnosis

are lacking. Therefore, a good marker should ideally not only indicate the

development of the disease, but also be able to differentiate RA from other

similar diseases and to help early diagnosis. The new marker which has

been tested in this study is the anti citrullinated protein antibody "ACCP2A".

Objective: To compare the diagnostic value and clinical utility of (ACCP2A)

with IgM rheumatoid factor (IgM RF) in patients with RA in Gaza Strip.

Methods: In cross sectional study; ACCP2A and IgM RF were determined in

142 serum samples; 67 patients suffering from arthritis, and 75 healthy

controls. All patients were diagnosed according to the guidelines of

American College of Rheumatology (ACR) and by rheumatologist. Both tests

were assessed by enzyme linked immunosorbent assay (ELISA). C-reactive

protein (CRP) and erythrocyte sedimentation rate (ESR) were also assayed

as inflammatory parameters for all subjects. All clinical features (onset for

RA, early symptoms, classification criteria, extra-articular disease, and

radiographic manifestations) and medications were investigated by

questionnaire interviews. And all results were analyzed by SPSS version

"15" .

Results: At a cutoff value of (>5 U/ml) for ACCP2A; sensitivity was 56% and

specificity was 99% and at a cutoff value of (>6 IU/ml) for IgM RF; it had a

higher sensitivity (85%) and a lower specificity (69%) than ACCP2A. The

area under the receiver operating characteristics (ROC) curve (AUC) for

ACCP2A was 0.80 (95% confidence interval, 0.70 – 0.90) and for IgM RF

was 0.83 (95% confidence interval, 0.74 – 0.92). In addition, there were

significant differences in the mean levels of ACCP2A (34.5 v 1.4) and IgM RF

(98 v 20) between RA patients and healthy plus other patient groups. Although

the concordance between both tests was significant (p= 0.000), IgM RF as the

single positive marker was found in 35% and ACCP2A as a single marker in 6%.

However, when the two markers were tested simultaneously, sensitivity and

specificity reached 88% and 100%. There was no significant correlation between

ACCP2A and personal characters such as age, gender, family history and

smoking. IgM RF didn’t show significant correlation with these characters except

with gender. A correlation with extra-articular manifestations, severity of

erosions and CRP was observed for both tests. ACCP2A didn't show significant

correlation with ESR but IgM RF showed a significant correlation with it.

Conclusion: ACCP2A was more specific than IgM RF in diagnosis of RA. So it

could be a useful serological assay in establishing the diagnosis of RA

especially in ambiguous cases or RF negative patients with RA. However, a

combination of both assays acts as "gold standard" in diagnosis of RA patients

in Gaza Strip. And both assays are also a better predictor of disease severity.

Keywords; Rheumatoid Arthritis, anti cyclic citrullinated peptide 2 antibody,

rheumatoid factor, Gaza Strip

III

مع عامل الروماتزم في تشخيص 2مقارنة مستوي األجسام المضادة ضد بروتين السيترولين التهاب المفاصل الروماتزمي في قطاع غزة

الخالصة

ة ر األمراض :قدم ن أآث اتزمي م اب المفاصل الروم ر الته ثيعتب يوعا حي ة ش وم المناعي ةيق از المناع جه

م ل جس ة المفاص ن بمهاجم تمر ع ث مس إن البح ذلك ف ه، ل دقيق لتشخيص د الفحص ال رض يفتق ذا الم و ه

ابهةحوصات جديدة تكون قادرة علي التعرف علي تطور المرض و علي تميزه عن األمراض األخر ى المش

ا في ه في األعراض بل ويكون قادرا أيضا علي تشخيصه مبكرا حيث إن التشخيص المبكر يعتبر عامال هام

.لم أمراض الروماتزم للبدء بالعالج مبكرا

روتين الس :هدف ام المضادة ضد ب ترولينيمقارنة القيمة التشخيصية و الفائدة الطبية لكل من فحص األجس

.وماتزم في مرض التهاب المفاصل الروماتزمي في قطاع غزةعامل الر

ة مصلية142 في ترولين وعامل الروماتزمياألجسام المضادة ضد بروتين الس تم فحص آل من :طريقة عين

مل ات تش ذه العين ات المفاصل و 67ه ن التهاب انون م ا يع ابطة 75 مريض ة ض ارهم آعين م اختي ع. ت جمي

ةمرضي تم تشخيصهم ي وضعتها الرابطة األمريكي ايير الت ا للمع إآلينيكيا بواسطة أخصائي الروماتزم وتبع

زا . م 1987روماتزم عام ة اإللي م أيضا. وقد تم إجراء التحاليل الخاصة بكال الفحصين بواسطة طريق ا ت آم

ي النشط اب البروتين ل االلته راء فحوصات عام دم الح" سي"ج رات ال اييرو سرعة الترسيب لك راء آمع م

. اللتهابات في آل عينة

ي وضعتها الرابطة ايير الت رة، المع دء المرض، األعراض المبك ل ب المرض مث ة ب م تقيم المعلومات المتعلق

م ائج ت تبيان وآل النت ة بواسطة اس رات اإلشعاعية و األدوي ألمريكية، مظاهر المرض خارج المفاصل، التغي

"SPSS "15حليلها باستخدام

يترولين هي : لنتائج روتين الس في المرضي وعدم انتشاره في% 56نسبة انتشار األجسام المضادة ضد ب

رول هي ي المرضي هي % 99كنت اتزم ف ار عامل الروم بة انتش رول% 85ونس ي الكنت اره ف دم انتش وع

ي %. 6 اس المساحة تحت منحن ا بقي ا قمن دنا أنROCولمعرفة القيمة التشخيصية لكال الفحصين فإنن فوج

اتزم هي0.80مساحة بالنسبة لألجسام المضادة ضد بروتين السيترولين هي والمساحة بالنسبة لعامل الروم

رول 0.8 . ، باإلضافة إلي وجود اختالف ذو داللة إحصائية في متوسط قيمة الفحصين بين المرضي و الكنت

ائية فإن وجود عامل الروماتزم لوحده في المرضيعلي الرغم من أن العالقة بين الفحصين لها داللة إحص

IV

ملل

ف

ل

ع

الو

ال

و

ال

لل

إ

ل

ت

ا

ت

ا ال

9

ال

3

و

رغم%. 6ووجود فحص األجسام المضادة ضد بروتين السيترولين لوحده آان % 35آان حوالي لكن علي ال

ات المرض في المرضي هي ات عدم% 88من ذلك فإن الدراسة أثبتت أن قدرة الفحصين معا علي إثب واثب

%.100وجودهم في الكنترول هي

ا يترولين وآم روتين الس ادة ضد ب ام المض ين األجس ائية ب ة إحص ود عالق دم وج ا ع ة أيض رت الدراس أظه

دخين ائلي، الت اريخ الع ر، الجنس، الت ل العم ببة للمرض مث اتزم. العوامل المس ا أن فحص عامل الروم و آم

. أعطي عالقة بالمتغيرات عدا عالقته مع الجنس

لوحظت" سي "ض خارج المفاصل وشدة تآآل المفاصل و البروتين النشط وعلي الرغم أن العالقة مع األعرا

ة إحصائية مع سرعة م يعطي عالق يترولين ل روتين الس مع آال الفحصين فإن فحص األجسام المضادة ضد ب

.الترسيب لكرات الدم الحمراء لكن فحص عامل الروماتزم أعطي عالقة إحصائية

ر فحص األجسام المضادة ضد :االستنتاج ر أآث ة بروتين السيترولين يعتب يدق اتزم ف من فحص عامل الروم

اتزمي اب المفاصل الروم خيص الته خيصو. تش ات تش ي إثب ة ف يرولوجية الهام ن الفحوصات الس ر م يعتب

اتزم المرض خاصة في الحاالت الغامضة أو المرضي الذين ي عامل الروم اؤهم عل و توصي. ال تحتوي دم

يحيث أن هذا يعتبر معا عمل الفحصين ب الدراسة اتزمي ف المعيار الذهبي في تشخيص التهاب المفاصل الروم

.قطاع غزة

V

Dedication

This thesis is dedicated to

Palestinian people

My fathers, pure soul

My family (mother, sisters and brothers)

VI

Acknowledgment

I wouId like to acknowledge all of the people who have assisted me with my

work presented in this thesis. My deepest gratitude goes to my supervisor,

Prof. Mohammad Eid Shubair, who has given me his utmost support,

guidance in all steps through this project.

I would also like to thank Dr. Mrwan El Hinde (head of rheumatism section

in EL SHIFA hospital), who helped me to recruit and assess the patients in

the study and for providing facilities for sample collection from them. So my

sincere thanks go out also to all of the patients that agreed to participate in

this study, without them this study would not have been possible.

I wish to gratefully acknowledge to the engineer Hani Mater for helping me

in buying and preparing the material and kits from outside of Gaza strip.

I am very grateful to Mr. Yosif El Arkan for helping me in the practical part

of research and for his permission for me to make part of work in his lab.

And I wish also to express my thanks to Mr. Naser Abu Shaban for his

useful advice in the practical part of research.

I owe my thanks to Dr. Samir Safi (Assistant Professor of Statistics) for

teaching and helping me in doing statistical analysis.

I want to mention with thanks Mr. Abdel Karim El Asele (technologist

assistant) for helping me in blood collection from healthy control.

I wish to present my thanks to the people who served as healthy controls in

my study.

In addition, I express my thanks to my aunt and Public Aid hospital for

preserving the samples during electricity cut off from the university.

Finally, I would like to thank my friends and relatives for their ongoing

support and encouragement.

VII

Items

Declaration Abstract (English) Abstract (Arabic) Dedication Acknowledgment Table of contents List of tables List of figure List of abbreviation

Chapter Chapter I Introduction 1.1 Overview 1.2 Objectives 1.3 Significance Chapter II Literature Revie2.1 Background 2.2 History 2.3 Epidemiology 2.4 Etiology 2.4.1 Genetic risk factors 2.4.1.1 HLA genes 2.4.1.2 Non HLA genes 2.4.2 Other risk factors 2.4.2.1 Gender 2.4.2.2 Smoking 2.4.2.3 Infection 2.4.2.4 Miscellaneous fac 2.4.3 Gene-environmental int2.5 Pathogenesis 2.6 Onset, course, clinical man 2.6.1 Onset and course 2.6.2 Non specific symptoms 2.6.3 Clinical manifestation 2.6.3.1 Articular manifesta 2.6.3.2 Extra-articular ma2.7 Mortality 2.8 Diagnosis 2.8.1 Diagnostic criteria 2.8.2 Laboratory diagnosis 2.8.2.1 Abs in the diagnos A. RF :A non specifi B. New markers spe C. ACCP2A: specifi

Table of Contents

Page

I II IV VI VII VIII XI XII XIV

1 1 4 4

w 5 5 5 6 8 8 8 9 9 10 10 10

tors 10 eraction 10

11 ifestation 16

16 16 16

tion 16 nifestation 18

20 20 20 22

is of RA 22 c Ab for RA 22 cific for RA 23

c Ab for RA 23

VIII

4.4 The concordance between ACCP2A and IgM RF 51

IX

D. Citrullination 24 E. Genetic association between citrullination and RA 25 F. ACCPA assays 27 G. Clinical utility of ACCP2A 28 H. Future trends of ACCP2A 31 2.8.2.2 Inflammatory markers in RA 32 A. ESR 32 B. CRP 32 C. The importance of ESR and CRP in RA 33 2.9 Imaging studies 33 2.10 Treatment 33 Chapter III Materials and Methods 35 3.1 Materials 35 3.1.1 Reagents kits 35 3.1.2 Instruments and disposable 35 3.2 Study population 35 3.2.1 Patients group 36 3.2.2 Healthy control 36 3.2.3 Questionnaire 36 3.2.4 Permission and ethical consideration 36 3.3 Methods 36 3.3.1 Sample collection 36 3.3.2 Sample processing 37 3.3.2.1 ACCP2A assay 37 A. Principle of assay 37 B. Kit components 37 C. Procedure (semi quantitative protocol) 38 D. Calculation of results 38 3.3.2.2 IgM RF assay 39 A. Principle of assay 39 B. Kit components 40 C. Procedure (semi quantitative protocol) 40 D. Calculation of results 40 3.3.2.3 CRP assay 41 A. Principle of assay 41 B. Kit components 41 C. Procedure (qualitative method) 41 D. Interpretation of result 42 3.3.2.4 ESR assay 42 A. Principle of method 42 B. Method components 42 C. Procedure 42 3.4 Statistical methods 43 Chapter IV Results 44 4.1 Characteristic of study population 44 4.2 Sensitivity and specificity of ACCP2A and IgM RF for diagnosis of RA 46 4.3 Comparison between the mean levels of ACCP2A and IgM RF in RA, controls plus others groups

48

4.5 Prevalence of ACCP2A and IgM RF in RA patients 51 4.6 prevalence of ACCP2A and IgM RF in controls group and others patients 52 4.7 A correlation of ACCP2A and IgM RF with inflammatory parameters (CRP and ESR) and disease activity parameters (radiological manifestation and extra-articular manifestation

53

4.8 Comparison between ACCP2A (+) and ACCP2A (-) 54 4.9 Relation of ACCP2A and IgM RF with personal characters of RA patients (age, gender, family history and smoking)

56

Chapter V Discussion 57 5.1 Diagnostic performance of ACCP2A 58 5.1.1 Sensitivity of ACCP2A and IgM RF 58 5.1.2 Specificity of ACCP2A and IgM RF 59 5.1.3 Sensitivity and specificity of combination (both or one) of ACCP2A and IgM RF

60

5.1.4 Comparison the diagnostic accuracy between ACCP2A and IgM RF 60 5.2 Diagnosis of sero-negative RA by ACCP2A 61 5.3 ACCP2A is predictive for the outcome of early RA 61 5.4 Use of ACCP2A to differentiate RA from other similar diseases 62 5.5 Prognostic ability of ACCP2A 63 5.6 Why ACCP2A RA patients have negative results? 65 5.7 Correlation of ACCP2A and IgM RF with personal characters of RA patients

67

5.8 Adopting model for lab diagnosis of RA in Gaza Strip 68 Chapter VI Conclusions and Recommendations 69 6.1 Conclusions 69 6.2 Recommendations 70 References 71 Annexes 98 Annex 1: RA patient's questionnaire 98 Annex 2: Approval to conduct the study from Helsinki committee in the Gaza strip

101

Annex 3: Approval to conduct the study in hospitals 102

X

List of tables

Table (2.1)……………………………………………………………………………………. Prevalence and incidence rates of RA worldwide (cases per 100 inhabitants)

7

Table (2.2)……………………………………………………………………………………. Main extra-articular manifestations of RA

19

Table (2.3)……………………………………………………………………………………. The 1987 criteria of ACR for RA

21

Table (2.4)……………………………………………………………………………………. The seven variables of a prediction model for persistent (erosive) arthritis

30

Table (4.1)……………………………………………………………………………………. Characteristics of RA patients

44

Table (4.2)……………………………………………………………………………………. Characteristics of non RA and UA patients

45

Table (4.3)……………………………………………………………………………………. Characteristics of controls group

45

Table (4.4)……………………………………………………………………………………. Diagnostic value of ACCP2A and IgM RF for RA

46

Table (4.5) …………………………………………………………………………………… Comparison between ACCP2A and IgM RF in RA patients, controls and other patients

50

Table (4.6) …………………………………………………………………………………… The relationship between ACCP2A and IgM RF

51

Table (4.7) …………………………………………………………………………………… Cross tabulation of ACCP2A and IgM RF in RA patients

51

Table (4.8) …………………………………………………………………………………… Prevalence of ACCP2A and IgM RF in controls group and others patients

52

Table (4.9)……………………………………………………………………………………. Correlation between Abs assay and inflammatory and disease activity parameters

53

Table (4.10)………………………………………………………………………………….. Comparison of patients who are ACCP2A (+) and who are ACCP2A (-) in relation to different variables

55

Table (4.11) …………………………………………………………………………………. Relation of ACCP2A and IgM RF with personal characters of RA patients

56

XI

List of Figures

Figure (2.1)………………………………………………………………………………….. Gene-environment interactions Figure (2.2)…………………………………………………………………………………..

Cytokine Signaling Pathways Involved in Inflammatory ArthritisFigure (2.3 a)…………………………………………………………………………………

Normal Knee JointFigure (2.3 b)…………………………………………………………………………………

Pathogenesis of early RA Figure (2.3 c)………………………………………………………………………………… Pathogenesis of established RA Figure (2.4)…………………………………………………………………………………..

Synovitis of index and long finger PIP joints Figure (2.5)…………………………………………………………………………………..

Swan neck and Boutonn- iere deformities of fingersFigure (2.6)…………………………………………………………………………………..

The distribution of RA in jointsFigure (2.7)…………………………………………………………………………………..

The masses on the dorsum of the hand are the ruptured tendonsFigure (2.8)…………………………………………………………………………………..

Rheumatoid nodules in a patient with long-standing RAFigure (2.9)…………………………………………………………………………………..

Patient with scleritis and scleral perforansFigure (2.10)…………………………………………………………………………………

Schematic presentation of IgM RFFigure (2.11)…………………………………………………………………………………

Citrullination (deimination) of peptidylarginine by PAD Figure (2.12)…………………………………………………………………………………

Possible links between RA specific ACCPAS and RA-associated genetic factors

Figure (3.1)…………………………………………………………………………………. Typical standard curve of ACCP2A

Figure (3.2)…………………………………………………………………………………. Typical standard curve of IgM RF

Figure (4.1)…………………………………………………………………………………. ROC curve of ACCP2A

Figure (4.2)…………………………………………………………………………………. ROC curve of IgM RF

Figure (4.3)…………………………………………………………………………………. Comparison between ACCP2A and IgM RF ROC curves

Figure (4.4)…………………………………………………………………………………. Test distribution of ACCP2A

Figure (4.5)…………………………………………………………………………………. Test distribution of IgM RF

XII

11 .

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15

. 17

. 17

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.

. 19

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. 39

. 41

. 47

. 48

. 48

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27

25

22

19

18

17

15

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14

Figure (4.6)………………………………………………………………………………….

Comparison between ACCP2A and IgM RF in RA patients, controls and others patients

Figure (4.7)…………………………………………………………………………………. Frequency of ACCP2A and IgM RF in RA patients

Figure (5.1)…………………………………………………………………………………. Presence of ACCP2A precedes the clinical onset of RA by years

Figure (5.2)…………………………………………………………………………………. Is it RA?

XIII

. 50

. 52

. 62

. 68

List of abbreviation Ab Antibody Abs Antibodies ACCP2A Anti Cyclic Citrullinated 2 Peptide Antibody ACR American College of Rheumatology AFA Anti Filaggrin Auto Antibodies Ag Antigen Ags Antigens AKAS Anti Keratin Antibodies APFS Anti Perinuclear Factor Antibodies ARA American Rheumatism Association AUC Area Under Curve CCPS Citrullinated Peptide Antibodies CI Confidence Interval CRP C-Reactive Protein DIP Distal Interphalangeal Joints DMARDS Disease Modifying Anti Rheumatic Drugs EBV Epstein Barr Virus ELISA Enzyme Linked Immuno Sorbent Assay ESR Erythrocyte Sedimentation Rate HCV Hepatitis C Virus HLA-DR Human Leukocyte Antigen-Determinants Region IL Interlukein JRA Juvenile Rheumatoid Arthritis MBP Myelin Basic Protein MCP Metacarpophalangeal MHC Major Histocompatibility Complex MRI Magnetic Resonance Imaging MTP Metaarsophalangeal NSAIDS Non Steroidal Anti Inflammatory Drugs OD Optical Density PAD Peptidyl Arginine Deiminase PIP Proximal Interphalangeal PMR Polymyalgia Rheumatism PR Palindromic Rheumatism PSA Psoriatic Arthritis PTPN22 Protein Tyrosine Phosphatase N22 RA Rheumatoid Arthritis RBCs Red Blood Cells RF Rheumatoid Factor ROC Receiver Operating Characteristics SE Shared Epitope SLE Systemic Lupus Erythematosus SNP Single Nucleotide Polymorphism TNF α Tumor Necrosis Factor α UA Undifferentiated Arthritis

XIV

Chapter I Introduction 1.1 Overview

Rheumatoid arthritis is a chronic inflammatory autoimmune disease

characterized by joint destruction, which leads to functional decline and disability

as well as increased mortality [1]. Although the overall prevalence of RA

approaches 1% among adults, like most autoimmune diseases, RA has a

predisposition for women, with a female to male ratio of 3:1 [2], the peak

incidence between 30 and 50 years [3].

RA is a multifactorial disease and its occurrence and severity are related to both

genetic and environmental factors [4]. The most important genetic association in

RA is with the HLA-DRB1 gene within the MHC, where particular alleles within

the DRB1*04 and *01 clusters encode the so-called SE sequences within the

expressed DRB1 molecule. Environmental factors include smoking and a

number of infectious diseases may be considered to be risk or precipitating

factors for RA [5].

The clinical course of RA is variable and its prognosis is difficult to predict. In

many patients, the disease process is severe and results in progressive joint

destruction and serious disability, but outcomes vary widely. Predicting the

outcome of this disease is crucial for optimal clinical management. Patients with

a high likelihood of an untoward outcome should be given appropriately

aggressive treatment at an early stage; this is even more important as

treatments have been shown to reduce progression of the disease. The ultimate

goal of treatment is remission that is, complete suppression of disease activity

[6].

1

A problem to be dealt with in the diagnostic research of RA is the lack of an

independent gold standard for the disease [7]. Doctors usually base the

diagnosis of RA on the presence of at least four parameters out of seven

approved by the (ACR);

1. Morning stiffness for at least one hour.

2. Soft tissue swelling (arthritis) of 3 or more joint areas PIP, MCP and MTP,

wrist, elbow, knee or ankle observed by a physician.

3. Swelling (arthritis) of the wrist, MCP or PIP areas.

4. Symmetric swelling (arthritis).

5. Rheumatoid nodules observed by a physician.

6. Presence of RF.

7. Radiographic changes (erosions and/or periarticular osteopenia) in the

hand, wrist, PIP or MCP areas [8]. However, these criteria were originally developed in an established patient

population to classify RA in order to be able to compare different patient

populations. So, these criteria are not the optimal instrument to distinguish early

RA from undifferentiated polyarthritis [9].

The first immune abnormality described in patients with RA was the production

of Abs, so-called “rheumatoid factors,” directed against the constant region of

IgG [10]. It is one of the seven classification criteria for RA proposed by ACR

[11]. While RF is elevated in ~75% of patients with RA [12], this autoantibody lacks

specificity. Sera from patients with other autoimmune disorders or infectious

diseases as well as up to 15% of the healthy, elderly population demonstrate the

presence of RF [13].

During recent years, various other circulating Abs have been reported to be of

potential diagnostic value, including APFs, AKAs, and anti-Sa Abs. Despite the

fact that several research groups have recognized the clinical and diagnostic

2

value of these Abs, especially because of their high specificity for RA, APFs and

AKAs and anti-Sa Abs never became very popular. This was mainly a

consequence of the fact that testing their presence was more laborious [14]. In 1998, Schellekens discoverd that all of the above Abs are likely directed

against target citrullinated proteins [15]. Citrulline is formed by the post-

translational modification of peptidylarginine by PAD. There are several isotypes

of this enzyme; in the inflammatory RA synovium, PAD 2 and PAD 4 are

abundant. These enzymes cause the local citrullination of synovial proteins,

such as fibrin [16]. Interestingly, citrullinated peptides fit better in the HLA–DR4

(DRB1*0401 or *0404) Ag binding grooves than the corresponding arginine

containing peptides, findings that link this immune response to the SE

hypothesis of RA pathophysiology. Citrullinated extracellular fibrin in the RA

synovium may be one of the major autoantigens driving the local immune

response suggested by the discovery of local production of ACCP2A and anti-

citrullinated filaggrin Abs in the joint. Also, functional haplotypes of PAD 4 may

be associated with RA.

Detailed studies of citrullinated filaggrin peptides showed that different patients

with RA recognized different linear citrullinated peptides, indicating a polyclonal

response. Flanking regions around the citrulline residue are important for the

reactivity, so not all sera are reactive with every peptide. The first generation of

ELISA for anti-CCP (CCP1), using several filaggrin epitopes, then various cyclic

epitopes that mimic true conformational epitopes were selected from libraries of

citrullinated peptides for the widely available 2nd generation anti-CCP assay

(CCP2) [17]. ACCP2A has high specificity (91–98%) but wide variability in

diagnostic sensitivity (41–68%) [18]. This research will focus on the efficacy of

ACCP2A as compared to IgM RF to assist physicians in the proper diagnosis of

RA in Gaza Strip.

3

1.2 Objectives

General objective Compare between ACCP2A and IgM RF in diagnosis of RA in Gaza Strip.

Specific objectives 1- Compare the sensitivity and specificity of ACCP2A and IgM RF for detection

of RA in Gaza Strip.

2- Evaluate the diagnostic of ACCP2A compared with IgM RF.

3- Study the relationship between ACCP2A and some of risk factors such as

age, gender, family history and smoking status.

1.3 Significance

Lab diagnosis of RA in Gaza strip is dependant only on RF which has

very low specificity for RA. So it is important to introduce more specific test such

as ACCP2A in routine practice. In addition to its high specificity, it can enable

clinicians to effectively distinguish RA patient from other RA resembling

diseases. It can predict the outcome of early RA as it has prognostic ability for

the future development of RA and assist in planning therapeutic strategy. It is

the first research in this field in Gaza strip according to the knowledge of

researcher.

4

Chapter II literature Review 2.1 Background

RA is a chronic inflammatory disorder that is characterized by polyarthritis

with often progressive joint damage and disability, immunologic abnormalities,

systemic inflammation, increased comorbidity, and mortality [19]. Recent studies

show that joint injury in RA patients' progresses within 2 years from onset and

aggressive treatments from the early stage can prevent the following

progression of the disease. Hence, the necessities of early diagnosis and early

treatment have been emphasized [20].

2.2 History The name of RA is derived from the Greek. Rheumatos means "flowing",

and this initially gave rise to the term 'rheumatic fever', an illness that can follow

throat infections and which includes joint pain. The suffix -oid means

"resembling", i.e. resembling rheumatic fever. Arthr means "joint" and the suffix -

itis, a "condition involving inflammation". Thus RA was a form of joint

inflammation that resembled rheumatic fever [21].

The term rheuma was introduced about 2000 years ago, to describe a

substance that flows. Lesions typical of RA in appearance and distribution have

been found in skeletal remains of Archaic Indians, suggesting that RA may have

existed in North America 3000 years ago. Guillaume Baillou (Ballonius; 1558-

1616) introduced the concept of rheumatism as a systemic musculoskeletal

syndrome in a work published posthumously in 1642. In 1880 Landré-Beeauvais

described in his thesis nine women having a joint disease that he designated

“goutte asténique primitive”. Although that thesis is usually regarded as the first

clinical description of RA, the clinical picture and disease course were described

more clearly by Brodie in 1819 and by Charcot in 1853. Garrod coined the term

5

“RA” in 1858, but that name was not officially adopted before 1922 in the U.K

and not until 1941 by the ARA. The discovery of RF (Waaler 1940) made it

possible to distinguish seropositive arthritis. In 1958, Ropes presented the ARA

criteria for the classification of RA. Those classification criteria have been

modified and the new set of criteria applied, was subsequently proposed by the

ACR, formerly ARA in 1987 [22].

2.3 Epidemiology

RA occurs two to four times more frequently in women than in men [23]. And occurs in all races and ethnic groups. Although the disease often begins in

middle age and occurs with increased frequency in older people, children and

young adults also develop it [24]. When it occurs in children it is called JRA [25]. RA is the most common inflammatory joint disease, with prevalence between

0.5% and 1% worldwide [26].

Several prevalence and incidence studies of RA have been reported during the

last decades, suggesting a considerable variation of the disease occurrence

among different populations. The majority of prevalence studies carried out in

Northern European and North American areas estimated a prevalence of 0.5–

1.1%. Studies from Southern European countries reported a prevalence of 0.3–

0.7%. Studies from developing countries also report a relatively lower

prevalence of the disease (between 0.1% and 0.5%). A higher prevalence has

been reported in certain Native Americans, and a very low frequency of RA in

some areas of rural Africa. The annual incidence rates of RA vary between 20

and 50 cases per 100,000 inhabitants in North American and North European

countries. There are only few studies from Southern European countries

indicating a relatively lower occurrence of the disease. There are no studies on

RA incidence from developing countries. Previous studies from Japan

suggested a relatively higher incidence of the disease, but all of them were

based on previous identification criteria, and recent data do not confirm this

6

picture. Table 2.1 summarizes data on RA prevalence and incidence for several

areas of the world and several countries [27].

Population Prevalence Incidence

USA (general population)

0.9 – 1.1 0.02 – 0.07 North American

USA (native Americans)

5.3 – 6.0 0.09 – 0.89

England 0.8 – 1.1 0.02 – 0.04 Finland 0.8 0.03 – 0.04 Sweden 0.5 – 0.9 Norway 0.4 – 0.5 0.02 – 0.03 Netherlands 0.9 0.05 Denmark 0.9

North Europe

Ireland 0.9 Spain 0.5 France 0.6 0.01 Italy 0.3 Greece 0.3 – 0.7 0.02

South Europe

Yugoslavia 0.2 Argentina 0.2 Brazil 0.5

South America

Colombia 0.1 Japan 0.3 0.04 – 0.09 China 0.2 – 0.3 Taiwan 0.3 Indonesia 0.2 – 0.3 Philippines 0.2

Asia

Pakistan 0.1 Egypt 0.2 Israel 0.3 Oman 0.4

Middle East

Turkey 0.5 Africa 0 – 0.3

Table 2.1 Prevalence and incidence rates of RA worldwide (cases per 100 inhabitants) [27].

7

2.4 Etiology The etiology of RA is not fully understood [28]. It is clear that both genetic

and environmental factors play important roles [29]. 2.4.1 Genetic risk factors

RA is a complex autoimmune disease with a strong genetic contribution

to its pathogenesis. Studies on twins have shown concordance rates between

12% and 15% in monozygotic twins compared to 4% in dizygotic twins.

Calculations based on these data have estimated an overall heritability of about

60%, indicating that genetic factors account for the majority of population

susceptibility to RA [30]. The strongest evidence for the influence of genetic

polymorphisms on risk of RA relates to HLA genes, non HLA genes as well as

environmental factors.

2.4.1.1 HLA genes

HLA-DRB1 genes are the best known genetic factor associated with RA

[30]. This association was observed over 30 years ago [31] although how this

contributes to the aetiopathogenesis of RA remains unclear. Studies in a wide

range of populations have revealed that a number of HLA-DRB1 alleles

(DRB1*0101, *0102, *0401, *0404, *0405, *0408, *1001 and *1402) are

associated with RA [32]. All these alleles share the conserved amino acid

sequence called SE. These SE residues constitute a part of the Ag -presenting

binding site. The SE hypothesis postulates that the SE motif itself is directly

involved in the pathogenesis of RA by allowing the presentation of a peptide to

arthritogenic T cells [33]. And it is associated with disease occurrence, severity

and outcome [34, 35]. The contribution of HLA to the overall genetic risk has been estimated to range

from 30% to 50%. These data suggest that non-HLA genes are involved in RA

susceptibility [36].

8

2.4.1.2 Non HLA-genes A number of studies have shown associations of non- MHC genes and

RA. For the most part, these have been of marginal significance and not

reproduced. Probably the most interesting, and certainly the most consistent

association of a non-MHC gene with RA are with PTPN22. The PTPN22 R620W

polymorphism is associated with RA only in those seropositive for RF in studies

from the U.S. and the U.K. and this association has been extensively confirmed

in other Caucasoid groups [37]. However, other studies showed association with

both sero-positive and sero-negative RA. And it has recently been found to be

associated with several autoimmune disorders such as type I diabetes, SLE,

Graves' disease and Hashimoto thyroiditis [38].

Several other polymorphisms have been described, but extended replication

studies are required before definite conclusions on their significance can be

reached [39]. Iwamoto and his colleagues showed a positive association

between SNP PAD 4 gene and RA not only in the Japanese population but also

in populations of European descent. This gene is expressed in synovial tissues

and is associated with levels of CCPs [40]. And others include TNF α gene

polymorphisms, IL-1 and IL-10 genes polymorphisms [41, 42]. 2.4.2 Other risk factors Although genetic factors are important in the development of RA, not all

those who are genetically susceptible develop the disease. Twin studies in the

UK and Australia have shown disease concordance rates in monozygotic twins

of between 15 to 21%. Earlier studies also showed only modest concordance for

Ab and immunoglobulin production within twin pairs. There is weak evidence;

however, of an increased concordance of RA, RF, and other Abs within spouse

pairs. These observations have encouraged the search for environmental

triggers [43].

9

2.4.2.1 Gender RA is more common in women than it is in men. Observations, such as

the levels of male sex hormones, especially testosterone, are reported to be

lower in men with RA, or that women who take the oral contraceptive pill are at a

reduced risk of developing RA, suggest that hormonal factors may play a role in

the development of the disease. Pregnancy has also been considered as a risk

factor for RA, but the studies show that the onset of RA is rare during

pregnancy, yet increases after delivery [44]. 2.4.2.2 Smoking

Epidemiological studies have suggested that smoking is an independent

risk factor for RA, particularly RF positive disease [45- 47]. Interestingly, a

recent study found smoking to be a predictor primarily in the subset of patients

with RA-associated HLA-DRB1 genotypes, indicating that genetic and

environmental factors could interact in predisposing to RA [48].

2.4.2.3 Infection There have been a large number of infectious agents that have been

implicated in RA, including EBV and parvovirus, as well as other agents,

including bacteria such as Proteus and Mycoplasma [49].

2.4.2.4 Miscellaneous factors

Exposure to silica, organic solvents and mineral oil, dietary factors such

as coffee or meat and blood transfusion has also been suggested as risk factors

[49-51]. 2.4.3 Gene–environment interaction

Gene–environment interaction has been shown for SE and smoking

regarding susceptibility to RF-positive RA, and the presence of anti-CCP Abs

(CCP+) correlates strongly with the presence of the SE. It has been

10

demonstrated that MHC II molecules expressing the SE can bind and present

citrullinated peptides to T cells, and that a combination of SE-positive (SE+) and

CCP+ is highly predictive of future RF-positive RA. Contrary to nonsmokers,

bronchoalveolar lavage cells from cigarette smokers have been reported to

express citrullinated Ags. It was also recently reported that smoking is a risk

factor to develop CCP+ RA only in SE+ patients. Taken together, these

observations suggest that immunization against citrullinated Ags in SE+

persons, possibly after triggering by nonspecific adjuvants such as cigarette

smoke, may be critical for the initiation of RA [52].

Figure 2.1 Gene-environment interactions [53].

2.5 Pathogenesis

Although the pathogenesis of RA is still unresolved [54], genetic studies

have pointed to the association of RA with the HLA–DRB1 alleles. The HLA–

DRB1 alleles are encoding for the SE. The SE hypothesis postulates that the SE

motif (a conserved amino acid sequence in the peptide binding pocket of the

DRB1 molecule) is directly involved in the pathogenesis of RA by allowing the

presentation of an antigenic peptide to T cells [55]. The Ag could be either an

exogenous Ag, such as a viral protein, or an endogenous protein. Recently, a

11

number of possible endogenous Ags, including citrullinated protein, human

cartilage glycoprotein, and heavy chain binding protein, have been identified

[56].

Ag-activated CD4+ T cells stimulate monocytes, macrophages, and synovial

fibroblasts to produce the cytokines IL -1, IL-6, and TNF α through cell-surface

signaling by means of CD69 and CD11 as well as through the release of soluble

mediators such as interferon- γ and IL-17. IL -1, IL-6 and TNF α which are the

key cytokines that drive inflammation in RA. TNF α and IL-1 stimulate synovial

fibroblasts, osteoclasts and chondrocytes that release matrix

metalloproteinases, in particular stromelysin and collagenases, are enzymes

that degrade connective-tissue matrix and are thought to be the main mediators

of joint damage in RA, Furthermore, TNF α and IL-1 inhibit the production of

tissue metalloproteinase inhibitors by synovial fibroblasts. Activated CD4+ T

cells express osteoprotegerin ligands that stimulate osteoclastogenesis, which

then leads to bone degradation. These activated macrophages, lymphocytes,

and fibroblasts, as well as their products, can also stimulate angiogenesis, which

may explain the increased vascularity found in the synovium of patients with RA.

Endothelial cells in the synovium are activated and express adhesion molecules

that promote the recruitment of inflammatory cells such as neutrophils into joints.

Neutrophils release elastase and protease, which degrade proteoglycan in the

superficial layer of cartilage. Early erosion of cartilage and bone is associated

with the formation of a proliferating pannus. Activated CD4+ T cells also

stimulate B cells through cell-surface contact and through the binding of αLβ2

integrin, CD154 (CD40 ligand), and CD28, to produce immunoglobulins,

including ACCP2A and RF. The precise pathogenic role of RF is unknown, but it

may involve the activation of complement through the formation of immune

Complexes [57- 59].

12

According to these events, RA is divided into two stages which are early and

established RA. In the normal knee joint, the synovium consists of a synovial

membrane (usually one or two cells thick) and underlying loose connective

tissue. Synovial-lining cells are designated type A (macrophage-like

synoviocytes) or type B (fibroblast-like synoviocytes). In early RA, the synovial

membrane becomes thickened because of hyperplasia and hypertrophy of the

synovial lining cells. This closely associated with the process of lymphoid

neogenesis. T cells (predominantly CD4+) and B cells (some of which become

plasma cells) infiltrate the synovial membrane. These cells are also found in the

synovial fluid, along with large numbers of neutrophils. In the early stages of RA,

the synovial membrane begins to invade the cartilage. In established RA, The

synovial lining cells proliferate in numbers and state of activation. The

proliferative “pannus” behaves as a locally invasive malignancy, burrowing into

and destroying articular cartilage and subchondral bone. The pannus consists of

both type A and type B synoviocytes and plasma cells [59, 60]. The

pathogenesis of RA is summarized in (Figures 2.2 and 2.3 a, b, c).

13

Figure 2.2 Cytokine signaling pathways involved in inflammatory arthritis [59]. Th2 denotes type 2 helper T cell, Th0 precursor of type 1 and type 2 helper T cells, and OPGL osteoprotegerin ligand.

14

Cartilage

Figure 2.3 a Normal knee joint [59].

Figure 2.3 c Pathogenesis of established RA [59]

Figure 2.3 b Pathogenesis of early RA [59]

15

2.6 Onset, course and clinical manifestation 2.6.1 Onset and course

The natural history of RA varies from self-limited, non-erosive to severe,

destructive disease. The onset may be acute, subacute or insidious, which is the

most common type. RA may begin as monoarthritis from one joint and then

gradually spread to other joints, or as polyarthritis [61]. 2.6.2 Non specific symptoms

RA presents in 60% of patients with fatigue, anemia, generaiized

weakness, anorexia and nonspecitic musculoskeIetal symptoms before the

appearance of synovitis [62].

2.6.3 Clinical manifestation The symptoms of early and established RA often differ. In addition, during

any stages of this condition, the symptoms can vary widely from person to

person [63]. 2.6.3.1 Articular manifestation

Physical findings in RA include symmetrical joint inflammation early in

the course of the disease and manifestations of progressive joint destruction

with chronic disease. Warmth, swelling, pain, reduced range of motion, and

palpable effusions characterise active synovitis (Figure 2.4). Classically, RA

causes synovitis in the (MCP) joints, (PIP) and (MTP) joints in a symmetrical

distribution. Synovitis may lead to hyperextension at the PIP joints and flexion at

the DIP and this is called swan neck deformities (Figure 2.5) [64, 65].

16

Figure 2.5 Swan neck and Boutonn- iere

deformities of fingers [65].

Figure 2.4 Synovitis of index and long

finger PIP joints [65]. RA commonly affects the feet, wrists, and knee, as well as the cervical spine,

glenohumeral joints, and hips. The distribution of disease in joint is shown in

Figure 2.6 [66].

Figure 2.6 The distribution of RA in joints [66]

17

.

Another key of articular feature is the presence of morning stiffness. Morning

stiffness can be defined as “slowness or difficulty moving the joints when getting

out of bed or after staying in one position too long, which involves both sides of

the body and gets better with movement.” Most patients with RA report some

degree of morning stiffness. However, morning stiffness, by itself, may not be a

good discriminator between the different arthritic conditions. [67].

Later in the course of disease radiographs may show joint space narrowing,

diffuse osteoporosis, and eventually marginal bone erosions. The destructive

changes occurring in joints affected by RA include degeneration of cartilage,

bony erosions, and destruction of ligaments and tendons (Figure 2.7). A further

element is pannus, an expanding mass of synovial lining cells that extends into

articular cartilage and bone and which is filled with inflammatory cells [68].

2.6.3. Main e

Figure 2.7 The masses on the dorsum of the hand are the ruptured tendons (marked with arrows) [65].

2 Extra-articular manifestation

xtra-articular manifestations of RA are summarized

18

in Table 2.2.

Table 2.2 Main extra-articular manifestations of RA [65, 68].

Organ Manifestation

Blood vessels Systemic vasculitis (small vessels) Lungs Pulmonary nodules

Heart Pericarditis Skin Subcutaneous nodules (rheumatoid nodules) [Figure 2-

8] Eyes Keratoconjunctivitis sicca, Episderitis and Sceleritis

[Figure 2-9] Felty’s syndrome neutropenia , splenomegaly, skin hyperpigmentation

bacterial infection and skin ulcers

Kidney Mesangial glomerulonephritis (GN), amyloidosis, tubulointerstitial nephritis and drug toxicity.

Nervous system Myelopathy, cord compression, Nerve entrapment, Mononeuritis multiplex.

Interstitial fibrosis

Figure 2.8 Rheumatoid nodules in a patient with long-standing RA [65].

Figure 2.9 Patient with scleritis and scleral perforans [65].

19

2.7 Mortality Mortality in RA has been reported to be higher in RA patients, mainly

attributed to extra-articular manifestations particularly cardiovascular disease

[69, 70]. Other comorbidities are infections [71] and lymphomas [72]. The

provision of treatment has been rarely considered a factor influencing mortality

until recently. However it should now be considered since more aggressive

therapy can affect outcome and there is evidence, for example, that

methotrexate therapy improves life expectancy in RA [73].

2.8 Diagnosis 2.8.1 Diagnostic criteria

RA, like all forms of inflammatory arthritis, has no single specific

pathognomonic sign, symptom or laboratory feature [74]. ACR presented a core

set of classification criteria for RA in 1987, patients fulfilling at least four of seven

of ACR criteria are classified as having RA. Criteria 1 through 4 must have been

present for at least six weeks. Patients with two clinical diagnoses are not

excluded. ACR criteria is demonstrated in Table 2.3 [8].

20

Table 2.3 Criteria of ACR for RA [8]. Criterion

Definition

1. Morning stiffness Morning stiffness in and around the joints, lasting at least 1 hour before maximal improvement.

2. Arthritis of three or more joint areas

At least three joint areas simultaneously have had soft tissue swelling or fluid (not bony overgrowth) observed by a physician. The 14 possible areas are the right or left (PIP), (MCP), wrist, elbow, knee, ankle and (MTP) joints.

3. Arthritis of hand joints At least one area swollen in a wrist, MCP or PIP joint.

4. Symmetry of arthritis Simultaneous involvement of the same joint areas (as defined in 2.) on both sides of the body (bilateral involvement of PIP, MCP or MTP joints is acceptable without absolute symmetry).

5. Rheumatoid nodules Subcutaneous nodules over bony prominences, extensor surfaces, or in juxta-articular regions, observed by a physician.

6. RF Detected by a method positive in less than 5% of normal controls.

7. Radiographic changes Radiographic changes typical of RA on posteroanterior hand and wrist radiographs, which must include erosions or unequivocal bony decalcification localized in or most marked adjacent to the involved joints (osteoarthritis changes alone do not qualify).

Although ACR emphasis features of chronicity, it has limited use in early

diagnosis [75]. Early diagnosis of RA is of particular importance, because joint

damage occurs early in the course of RA; 30 % of patients have radiographic

evidence of bony erosions at the time of diagnosis, and this proportion increases

to 60 % by two years. Unfortunately, bony erosions and deformities are largely

irreversible. Initiation of therapy with DMARDs within three months after the

diagnosis of RA is crucial; a delay of as little as three months in the introduction

of these medications results in substantially more radiographic damage at five

years. Therefore, early diagnosis, although challenging, is critical [76].

Furthermore, the criteria may be present in some people in the early stages of

21

other rheumatic conditions [77, 78]. However rheumatologists are still used as

a ‘gold standard’ for diagnosis of RA [79]. 2.8.2 Laboratory diagnosis 2.8.2.1 Abs in the diagnosis of RA

The serum of RA patients contains a variety of Abs directed against self-

antigens. The most widely known of these Abs is the RF A. RF: A non specific Ab for RA

RF was first described about 75 years ago and to date, it is the only

serologic parameter that is included in ACR criteria [80]. RF is an Ab directed

against the Fc portion of IgG molecules [81] and found in every immunoglobulin

subclass (IgM, IgA and IgG) [82]. With the IgM class being the most common

[83]. (Figure 2.10).

The potential physiological role of IgM RF

includes the following:

1. The IgM bound on the surface of B

lymphocytes enhances Ag

presenting efficiency of these cells

for the Ag complexes with IgG and Figure 2.10 Schematic presentation

of IgM RF [84]. 2. Secreted IgM stabilizes low affinity

IgG-Ag complexes

o removes immunocomplexes via complement activation

o improves opsonization [84]. Although reasonably sensitive (60-80%) [85], the specificity of RF in RA is low

(70-80%) [86,87]. Around 80% of RA patients become IgM RF positive, though

this can take many years to occur. In other words, IgM RF has low sensitivity in

the early stages of RA. Furthermore, patients with other inflammatory diseases

(including Sjögren’s syndrome, SLE, chronic active hepatitis and bacterial

22

infections) are associated with a broadly elevated level of gamma globulin. RF is

also found in low titre (5%) in normal individuals, with titre and incidence

increasing with age. Thus RF has a relatively low specificity for RA [88,89]. This

has stimulated a search for novel Abs and their respective target molecules that

could be useful for the diagnosis of RA [90]. B. New markers specific for RA

Because RF has a limited specificity for RA, other more specific Abs have

been sought. The first such Ab was APF described by Nienhuis and Mandema

as early as 1964. This Ab directed against keratohyaline granules surrounding

the nucleus of human buccal mucosa cells [91]. In 1979 other Abs against Ags

of the corneal layer of the rat oesophagus were discovered and were called AKA

[92]. Nevertheless, although these Abs are increasingly considered as useful

diagnostic and, even, prognostic markers, their detection remains restricted to

specialised laboratories, essentially because of technical difficulties [93]. Recently it was shown that both APF and AKA belong to a single family of Abs

that target isoelectric variants of an epithelial protein, filaggrin, or of its

precursor, profilaggrin. These Abs were then considered as AFA [94]. C. ACCP2A: specific Ab for RA.

In 1998, Schellekens discovered that all of the above Abs recognize

epitopes containing non -standard amino acid citrulline [15]. Citrulline; the

naming of the Ab is simply determined by the method used to detect them. With

the most sensitive assay (CCP2), the relative amounts of ACCPAS in synovial

fluid and in extracts of synovial tissue of patients with RA is 1.4- and 7.5-fold

higher, respectively, than in sera of the same patients. Furthermore, ACCPA

secreting cells are found in the inflamed RA synovia. These data suggest the

presence of citrullinated proteins in the RA synovium causing an Ag driven

maturation of CCP-specific B cells at the site of inflammation [95]. Studies by

the group of Serre from France have clearly indicated that citrullinated

23

extracellular fibrin in the RA synovium may be one of the major autoantigens

driving the local immune response, and this is supported by the discovery of

local production of anti-citrullinated peptides/ protein Abs in the joint [96].

D. Citrullination Because citrulline, the antigenic determinant for these Abs, is a

nonstandard amino acid, it is not incorporated into proteins during translation. It

can, however, be generated post-translationally by enzymatic citrullination

(deimination) of arginine residues. This conversion is catalyzed by the enzyme

PAD (Figure 2.11) [97]. Five, highly conserved isotypes of PAD enzymes exist

in all mammalian species (PAD1–PAD4 and PAD6). The main difference

between the isotypes is their tissue-specific expression. For RA, PAD2 and

PAD4 are the most interesting enzymes as both of them are expressed in the

synovium and central nervous system. All PAD enzymes require high calcium

levels for activity (about 10-5 mol/l). Because the intracellular calcium

concentration in healthy cells is much lower (10-7 mol/l), the enzymes are only

activated during events that lead to high calcium levels, like cell death, or late in

the differentiation of skin cells (PAD1 and PAD3). Citrullination thus is believed

to have an important role in several physiological processes, including the

normal development of the myelin sheath and epidermis. Whereas arginine is a

positively charged amino acid, citrulline is uncharged, and citrullination of

proteins thus leads to a loss of net positive charge. As a result, the target

proteins adopt an altered, generally more open structure. This change in

structure may severely hamper the cellular function of these proteins.

Interestingly, all known natural substrates of PAD enzymes are proteins that

have an important structural function (for example, intermediate filament

proteins, MBP, histones, keratins, fibrin clots) [98].

24

Figure 2.11 Citrullination (deimination) of peptidylarginine by PAD [99].

E. Genetic association between citrullination and RA

Several genetic risk factors for severe RA might be functionally linked to

the production or effects of ACCPAs.

1. genetic polymorphisms in the PAD4 gene was associated with susceptibility

for RA. Although it was not shown whether the PAD4 encoded by the

susceptible haplotype exhibits an altered enzymatic function, it was shown that

the RA-susceptible haplotype increases PAD4 mRNA stability. In theory, this

could result in more PAD4 enzyme being produced and subsequently lead to

increased citrullination of proteins and a higher chance of developing ACCPAs

(Figure 2.12 a) [100]. 2. Correlation between RA and certain HLA haplotypes (e.g. HLA-DR4 [HLA-

DRB1*0401 and HLA-DRB1*0404]) has been known for more than 25 years

[101]. Recent studies indicate that HLA-DRB1*0401 major MHC molecules have

a high affinity for negatively charged or uncharged polar amino acids (e.g.

citrulline) whereas positively charged amino acids (e.g., arginine) inhibit peptide

binding. Because amino acid interactions at HLA anchoring pockets are not only

dependent on the charge of the residue but also the size, SE was large enough

to accommodate the side chain of citrulline (Figure 2.12 b). This citrulline-

specific interaction might be the basis of a citrulline- specific immune response.

It can cause a 100-fold increase in affinity for MHC with the SE. This could result

25

in a higher density of peptide-HLA complexes on Ag presenting cells, which may

exceed the biochemical margin of safety necessary for T cell proliferation and

activation [102]. Although there is no absolute requirement for HLA-DR4 in

order to develop ACCPAs, there is a strong correlation between HLA-DR4

status and ACCP positivity in RA patients [103]. 3. SNP in the IL-10 promoter is associated with high IL-10 production and this

will enhancing B-cell proliferation, differentiation and Ab production such as

ACCPA. ACCPAS will form immune complexes which can be bound by

inflammatory cells via their Fcγ receptors. This will activate these cells and

cause the release of extra proinflammatory cytokines (Figure 2.12 c). There are

also various polymorphisms in proinflammatory cytokines and their receptors are

thought to be associated with RA (Figure 2.12 d). These genetic factors cause

the release of larger amounts of cytokines upon stimulation or cause cells to be

more sensitive towards these cytokines. The cytokines are the motor of the

inflammation, causing influx and activation of more inflammatory cells. These

cells will eventually die, allowing their PAD enzymes to become activated by

influxing Ca2+. With this, the cycle is complete and will continue if not stopped.

The cycle will ultimately lead to the chronic inflammatory disease we call RA.

Besides these genetic factors, other susceptibility loci might also be involved.

Their precise nature needs to be clarified in order to understand their possible

role in the triggering or progression of RA [104].

26

Figure 2.12 Possible links between RA specific ACCPAS and RA-associated genetic

factors [104]. DC, dendritic cell; Fcγ, Fcγ receptor; IC, immune complex; mø,

macrophage. F. ACCPA assays

The citrulline moiety of the autoantigens mentioned previously is the

essential determinant on proteins recognized by APF, AKA and anti-Sa [105]. From the detailed studies of citrullinated filaggrin peptides by Schellekens, It

became apparent that different RA patients recognise different linear citrullinated

peptides, which would indicate a polyclonal response against probably many

Ags. The citrulline-flanking residues influence the reactivity with the Abs. In the

first attempts to design an ELISA for detecting ACCPS several linear filaggrin

epitopes were used. The sensitivity of this initial ELISA could be improved by

making cyclic variants of peptides in which the citrulline residue is optimally

exposed for Ab binding. This led to the first generation anti-CCP (cyclic

citrullinated peptide) test (named CCP1 test). All studies undertaken using this

27

assay agreed that this assay displayed a high diagnostic specificity with a

nosographic sensitivity of around 65%. However, the idea that filaggrin is most

likely not the triggering agent in the ACCPS response, since it is not present in

the joint, sparked investigations to obtain novel peptides from dedicated libraries

of citrullinated peptides using RA sera for selecting the most reactive species.

After selection, such peptides were made cyclic to ensure exposure of the

antigeneic citrulline moiety. These investigations have resulted in the second

generation anti-CCP assay, which is now available world-wide as the ACCP2A

test [106].

G. Clinical utility of ACCP2A 1. Use in diagnosis of RA

The most important requirements for a good diagnostic marker are that it

must be specific for the disease and present in a relatively high percentage of

patients. Several studies using the ACCP2A test show that this test indeed

provides a good sensitivity (41–80%) and excellent specificity (89–100%) [107]. Many studies have verified the validity of the ACCP2A assay and found that it

has a similar level of sensitivity for RA as the test for RF, i.e. approximately

80%. However, the ACCP2A assay has a far superior level of specificity, making

it a more ideal marker than RF. In fact, 40% of RF seronegative patients with

clinical signs and symptoms of RA are ACCP2A positive. Therefore, the

combination of the two tests greatly enhances the diagnostic accuracy of the

disease. Both are used in clinical practice routinely today [108]. 2. Use in diagnosis of early RA

The current therapeutic strategy uses increasingly aggressive regimens

early in the course of the disease as most of bony damage occurs in first two

years in 90% of patients. Thus, early diagnosis is crucial. The 1987 ACR criteria

are rarely met during the first few months of the disease. In many early cases of

28

RA, clinical symptoms are milder and nonspecific, and patients will not fulfill

ACR classification criteria for RA. Therefore, the detection of a disease specific

autoantibody like ACCP2A is important. ACCP2A may be detected in roughly

50-60% of patients with early RA usually after 3-6 months of symptoms. The

specificity of ACCP2A is around 95-98% as regards undifferentiated forms of

arthritis that do not develop into RA. IgM RF is often found in the same patients,

but with much lower specificity for RA. ACCP2A may pre-date arthritis by

several years. A study using the ACCP2A assay found progression from

undifferentiated polyarthritis to RA in 93% of ACCP2A positive patients but only

in 25% of ACCP2A negative patients after 3 years of follow-up [109].

3. Use in prognosis of RA Many studies support the idea that RA patients positive for ACCP2A

develop significantly more severe radiological damage than ACCP2A negative

patients. Recent studies using the ACCP2A test have confirmed the prognostic

value of the ACCP2As. Kroot and his colleagues assessed ACCP2A at baseline

in 273 patients and measured radiological joint damage (Larsen score) and

progression after six years follow-up. They found that patients with ACCP2A had

developed significantly more severe radiologic damage after 6 years of follow up

[110]. Another study indicate that the combination of ACCP2A and IgM RF

increased the ability to predict erosive and progressive disease [111].

In 2002 Visser and his colleagues [112] have developed a diagnostic model that

predicts three forms of arthritis outcome: self-limiting, persistent non-erosive and

persistent erosive arthritis. It consists of seven variables; these variables are

very similar to those that comprise the ACR. The corresponding weighting

system gives the prognostic capacity. The unusual addition to the other factors

is the ACCP2A (Table 2.4).

29

Table 2.4 The seven variables of a prediction model for persistent (erosive) arthritis [112].

Criterion

1. Symptom duration ≤6 weeks <6 months ≥6 months 2. Morning stiffness ≥1 hour 3. Arthritis ≥3 joint groups 4. Bilateral compression pain MTPs 5. IgM RF ≥5 IU 6. Anti-CCP ≥92 IU 7. Erosions X-rays hands or feet

These predictive values form a valuable basis from which therapeutic decisions

can be made in an early phase of the arthritis. The early recognition of persistent

(erosive) arthritis allows early intervention with DMARDs, which will lead to

earlier disease control and improvement of disease outcome. Otherwise, early

recognition of self limiting arthritis will prevent the unnecessary treatment of

these cases with potentially toxic DMARDs [112].

4. Use in differentiation of RA from other diseases Several studies show that the ACCP2A test enables clinicians to

effectively distinguish RA patients from other RA-resembling diseases, even in

cases where the RF is not discriminative. For example, a significant proportion

of patients with chronic HCV infection suffer from a symmetric inflammatory

polyarthritis that closely resembles the symptoms of RA. Since the majority of

these patients are RF positive, RF cannot be used to discriminate HCV-

associated arthritis from RA. The ACCP2A positivity in this group of patients,

however, is negligible compared to that in RA patients [113,114]. Also when

SLE is associated with polyarthritic phenomena, it can be confused with RA.

Mediwake and coworkers studied a large cohort of SLE patients, ten of whom

had erosive joint disease. Of these ten patients six were positive for RF,

whereas only 2 were ACCP2A positive. Of the non-erosive SLE patients, only

30

0.5% was ACCP2A positive, versus 18% for RF [115]. All these studies confirm

that the ACCP2A test indeed is an important and specific diagnostic tool for RA

and related disorders.

5. Use in determination of drug strategy There are some studies talking about the importance of ACCP2A in

determination of drug strategy. One of these for Alessandri and his colleagues

who indicate that anti TNF α treatment in RA results in a decrease in the serum

titers of RF and ACCP2A in patients showing clinical improvement, suggesting

that these measurements may be a useful adjunct in assessing treatment

efficacy [116]. H. Future trends of ACCP2A

• ACCP3A

While the majority of currently available ACCP2A assay is based on one

particular manufacturer's assay (for patent reasons), for increase the

sensitivity of ACCP2A, other manufacturers are actively involved in

developing their own ACCP assays (likely to be marketed as 'third or

subsequent' generation assays) [117,118].

• Microarray technique

The challenge to develop new specific markers is being met, as

demonstrated by the development of novel serologic assays with high

specificity being combined with other less specific Abs but when used

together yields test results that are virtually 100% specific for RA. This

developed and applied by William Robinson. He has applied the microarray

technique to profile autoantibody responses in cohorts of patients with RA

and control patients. In addition to demonstrating that autoantibody targeting

of citrullinated autoantigens is sensitive and specific for RA, patients with RA

with ACPA responses frequently exhibit autoantibody reactivity against

multiple citrullinated peptides and/or proteins contained on the arrays which

31

including citrullinated fibrinogen, citrullinated vimentin, citrulline-substituted

filaggrin peptides, heterogenous nuclear ribonucleoprotein particles, heavy

chain binding protein, type II collagen, and several heatshock proteins. This

technology is currently being applied to early diagnosis of RA [119,120]. If these new assays can be developed in a cost-effective manner with good

sensitivities, utilization of such assays could reliably diagnose rheumatoid

disease and allow treatment to proceed in an unambiguous fashion. For now,

the only available commercial assay is ACCP2A [121]. 2.8.2.2 Inflammatory markers in RA

While multiple blood markers of inflammation have been identified and

shown to be useful in the evaluation and treatment of RA, to date, ESR and

CRP have been most commonly studied and used in clinical practice.

A. ESR

The ESR, an indirect assessment of inflammation, measures the distance

that RBCs fall in a capillary tube over the course of an hour. The presence of

inflammation causes the cells to fall more quickly due to the action of

inflammatory proteins, such as fibrinogen or immunoglobulins, blocking the

normal charge inhibition on RBCs. In many RA studies, an ESR level greater

than 20 to 30 mm/h has been considered abnormal; however, considerable

individual variability between normal and abnormal tests exists [122].

B. CRP CRP is a pentameric protein released in response to inflammatory stimuli.

Because testing directly measures this protein, CRP levels are a more accurate

measure of inflammation than the ESR. Measuring CRP in inflammatory

conditions is preferred over the ESR as CRP responds much more quickly to

inflammatory stimuli and can, therefore, be used as a timely marker of active

inflammation. The CRP level that has been determined to be abnormal in RA

32

studies is generally greater than 1.0 mg/dL. But caution must be used when

interpreting this value as many clinical labs report CRP in mg/L, resulting in a

10-fold higher value that may still be a normal result [122]. C. The importance of ESR and CRP in RA.

Elevation of ESR and CRP are the strongest predictors of persistent,

progressive disease in RA. If they are elevated in the early disease and don't

improve with therapy, this will lead to joint damage and other worse outcomes

[123,124]. ESR and CRP are very sensitive to change in disease activity. ESR

becomes more than 50 mm/hr and CRP becomes more than 3 mg/dL. But the

levels of them decreased rapidly, beginning in the first month after the injection

of etanercept, and these reductions appeared to be sustained throughout the

etanercept treatment. Similar results were observed with another TNF α blocker,

infliximab [125,126]. In addition, if use of one anti TNF α agent does not result

in a decreased CRP level at 12 weeks, it may be an indication to use another

biologic agent [127].

2.9 Imaging studies X-ray, computerized tomography or MRI scans and other forms of

imaging can show damage to articular surfaces, and are used to measure the

degree of destruction of the joints and articular cartiIage and inflammation.

However, the usefulness of these techniques is limited to the later stages of the

disease, when the progression of RA has already led to the destruction of the

joints [61].

2.10 Treatment The goals of treatment in RA are to control inflammation, prevent

progressive joint destruction, preserve and improve activities of daily living, and

alleviate pain. Medical treatment includes the use of NSAIDs, DMARDs,

corticosteroids, and biological response modifiers. In addition to drug therapy,

33

nonpharmacological treatment, including patient education, physiotherapy,

occupational therapy, orthotics, and surgery are important.

A major transformation in the pharmacological treatment of RA occurred

in the 1990s. The previous therapeutic approach, termed as the therapeutic

pyramid, generally involved initial conservative management with NSAIDs for

several years; DMARDs were withheld until clear evidence of the progressive

character of the disease in the face of erosions was seen. DMARDs were then

added individually in slow succession if the disease progressed. This form of

treatment has been replaced by a) early initiation of DMARDs and b) use of

combinations of DMARDs in patients with the potential for progressive disease.

The idea of early intervention with DMARDs has been validated in a randomised

trial. Methotrexate has become the most widely prescribed DMARD. Other

commonly utilized DMARDs include leflunomide, sulphasalazine, and

hydroxychloroquine. Also cyclosporine, azathioprine, D-penicillamine, and gold

salts remain in use. DMARD monotherapy is often only partly effective and

poorly tolerated in long term. DMARD combination therapy has been explored

as a way to maximize therapeutic effect while maintaining a tolerable toxic-

effect profile. Initial randomized controlled trials of monotherapy versus

combination therapy yielded conflicting results. However, more recently it has

been shown that combination therapy has clear benefits and tolerable toxic

effects in both early and chronic RA. In general, the beneficial combinations

have included methotrexate and 1 to 3 other DMARDs [128-130].

In recent years, new biological drugs, especially TNFα blockers include

infliximab and etanercept, have been introduced into clinical use. In combination

with methotrexate, they have been able to retard considerably or even stop the

radiographically detected destruction of joints in RA [131,132].

34

CHAPTER III Materials and Methods 3.1 Materials 3.1.1 Reagent kits Two ELISA kits for ACCP2A assay

Two ELISA kits for IgM RF assay

Two CRP kits

3.1.2 Instruments and disposables

Microplate ELISA reader (TC 89 +)

Microplate ELISA reader (Tecan reader)

Alcohol swab Syringes and needles

Plasters

Chemistry tubes

Sodium citrate tubes (ESR tubes)

Distilled water

Micropipettes 10µl -1000µl

Yellow and blue tips

Eppendrof tubes

Timer

Centrifuge

Ice box for keeping the sample through transportation.

Deep freezer (-72°C) for specimen storage

3.2 Study population This study is across sectional study. Our goal was to recruit 150 subjects

divided equally into patients and healthy controls. However, due to different

problems we recruited only 67 patients and 75 healthy controls. So the total

35

number of individuals included in this study was 142 and were divided into the

following two groups:

3.2.1 Patients Sixty seven patients were suffering from arthritis and who were randomly

selected from the outpatient rheumatology unit at EL SHIFA hospital.

3.2.2 Healthy controls

Blood samples from healthy controls were collected from 75 blood donor

volunteers who had no records of arthritis.

3.2.3 Questionnaire All patients were questioned about their personal character, clinical

features (onset for RA, early symptoms, classification criteria, extra-articular

disease, and radiographic manifestations) and medications received. Patients

were also questioned about their ability to perform daily activities [133]. Most questions were yes/no and multiple choice questions (Annex 1). The

validity of the questionnaire was tested by three specialists. The questionnaire

was piloted. All interviews were conducted face to face by the researcher

herself.

3.2.4 Permissions and ethical consideration

According to research ethics, Permission was obtained from the Helsinki

Committee for sample collection and consents were also obtained from each

patient for the purpose of the study and drawing extra blood (Annexes 2 and 3).

3.3 Methods

3.3.1 Sample collection Five ml venous blood was collected from each subject, 1 ml was

delivered into ESR tube and the rest of the sample was delivered into a tube

36

without anticoagulant. The ESR tube was left for 1 hr at room temperature to

measure ESR (mm/hr). The second tube was centrifuged and then the serum

separated and stored in a -72°C freezer until used for ACCP2A assay, IgM RF

assay and CRP.

3.3.2 Sample Processing 3.3.2.1 ACCP2A assay ACCP2A levels were determined using DIASTAT TM (Axis-Shield com – UK)

A. Principle of the assay The wells of the microtitre strips were coated with a highly purified

synthetic cyclic peptide containing modified arginine residues. During the first

incubation, specific autoantibodies in diluted serum or plasma bind to the Ag -

coated surface. The wells were then washed to remove unbound components.

In the second incubation, the conjugate, an enzyme-labelled monoclonal Ab to

human IgG, binds any surface-bound autoantibodies. After further washing,

specific autoantibodies were traced by incubation with the substrate. Addition of

stop solution terminates the reaction, resulting in a coloured end-product. The

amount of conjugate bound was measured in absorbance units. The

concentration of anti-CCP autoantibody was estimated by interpolation from a

dose-response curve based on standards.

B. Kit components Conjugate, substrate, stop solution, wash buffer, CCP-coated wells and

strip holder, sample diluent, 5 ACCP standards with concentrations (0 U/ml, 2

U/ml, 8 U/ml, 30 U/ml and 100 U/ml), ACCP reference control and positive and

negative controls.

37

C. Procedure: (Semi quantitative protocol) 1. Allow all kit components, including the microtitere strips, to warm up to 18-

25°C for 30-60 minutes before use. Mix reagents by gentle inversion.

2. Dilute the following reagents before use and mix thoroughly; 1 vial of wash

buffer concentrate with 375 ml distilled water, 1 vial of diluent concentrate with

100 ml distilled water and 10µl of positive and negative controls with 1 ml diluted

sample diluent.

3. Reference wells for identification.

4. Pipette 100µl reference control/standards in duplicate, pre diluted (1:100)

positive and negative controls, and pre-diluted (1:100) patient samples into

appropriate wells.

5. Incubate for 60±10 minutes at 18-25°C.

6. Decant strip contents by quick inversion over a sink suitable for the disposal

of biological material, bearing in mind the potential infective hazard of the

samples. Blot inverted strips well with paper towels.

7. Wash wells three times with a minimum of 200µl wash buffer. Decant and blot

after each wash steps.

8. Add 100µl conjugate to each well.

9. Incubate for 30±5 minutes at 18-25°C.

10. Repeat steps 6 and 7.

11. Add 100µl substrate to each well.

12. Incubate for 30±5 minutes at 18-25°C. Don’t decant.

13. Add 100µl stop solution to each well. In the same order and rate as

substrate. Tap wells gently to mix. 14. Read strips within 24 hours at 550nm (540-565nm).

D. Calculation of results Plot the standard concentration on X-axis and plot the mean absorbance

value of each standard on Y-axis on suitable graph paper. Concentrations of

controls and samples can then be read from the standard curve (Figure 3.1).

38

The reference range of ≤ 5U/ml is considered negative and > 5U/ml is

considered positive.

0.0

0.4

0.8

1.2

1.6

2.0

2 8 30 100Anti-CCP (U/ml)

Abs

orba

nce

at 5

50nm

Figure 3.1Typical standard curve of ACCP2A

3.3.2.2 IgM RF assay IgM RF level was determined by using IgM RF kit (GA Generic

Germany).

A. Principle of the assay The RFs of standards, control and diluted patient samples

immobilized on the solid phase of microtiter plates. Following

period of 30 min at 37°C, unbound serum components were

washing step. The bound IgM Abs react specifically with conju

incubation period of 30 min at 37°C. Exessive conjugate is sep

solid phase immune complexes by the following washing s

converts the colorless substrate into a blue product. The enzy

stopped by dispensing a stop solution into the wells after 15 min

the solution from blue to yellow. The OD of the solution at 450

proportional to the amount of specific Abs bound. The concentra

39

Assays GmbH-

react with IgG

an incubation

removed by a

gate within the

arated from the

tep. Conjugate

me reaction is

at 37°C turning

nm is directly

tion of IgM RF

can be estimated by interpolation from a dose-response curve based on

Standards.

B. Kit components Microtiter plate, concentrated wash buffer, sample diluent, conjugate,

substrate, stop solution, 5 standards with concentrations of (6 IU/ml, 12 IU/ml,

50 IU/ml,100 IU/ml and 200 IU/ml) and positive control.

C. Procedure: (Semi quantitative protocol) 1. Bring all reagents to room temperature (18-25°C) before use. Mix gently

without causing foam.

2. Dilute patient sera with sample diluent 1:101

3. Prepare a sufficient amount of wash solution by diluting the concentrated

wash buffer 10 times with distilled water.

4. Dispense 100µl of standards (1-5), positive control and diluted patient

samples into the respective wells.

5. Seal plate; incubate 30 min at 37°C.

6. Decant, then wash each well three times using 300µl wash solution.

7. Add 100µl of substrate to each well

8. Seal plate; incubate 30 min at 37°C.

9. Decant, then wash each well three times using 300µl wash solution. 10. Add 100µl of substrate to each well.

11. Incubate 15 min protected from light at 37°C.

12. Add 100µl of stop solution to each well and mix gently.

13. Read the OD at 450nm versus 620 or 690nm within 30 min after adding the

stop solution.

D. Calculation of results The standard curve (Figure 3.2) is established by plotting the mean OD-

values of the standards 1-5 on the ordinate, Y-axis (lin. scale) versus their

respective IgM RF concentrations on the abscissa, X-axis, (log. Scale). IgM RF

40

concentrations of the unknown samples are directly read off in IU/ml against the

respective OD values. The reference range (<6) is considered negative, while

(>6) is considered positive.

IgM RF (IU/ml)

OD

450

nm

Figure 3.2 Typical standard curve of IgM RF

3.3.2.3 CRP assay CRP level were determined by using CRP latex kit (plasmatic- UK).

A. Principle of the assay Latex particles coated with goat anti-human CRP Abs are

when mixed with samples containing CRP.

B. Kit components CRP latex reagent, positive control, negative control, gly

buffer, pipette, stirrers and reusable agglutination slide.

C. Procedure (Qualitative method) 1. Allow each component to reach room temperature.

2. Gently shake the latex reagent to disperse the particles.

3. Place a drop of undiluted serum onto the circle of the test slid

disposable pipettes provided.

4. Add one drop of the latex reagent next to the drop of serum.

41

agglutinated

cine diluent

e using the

5. Using the other end of the pipette (broad end) spread the reagent and serum

sample over the entire area of the test circle.

6. Gently tilt the test slide backwards and forwards approximately once every

two seconds for two minutes. Positive and negative controls should be included

at regular intervals. Both are ready for use and do not require further dilution. At

the end of the test rinse the test slide with distilled water and dry. Normal

laboratory precautions should be maintained whilst handling patients, samples.

D. Interpretation of result Presence of agglutination indicates a level of CRP in the sample which is

≥ 6mg/l. The lack of agglutination indicates a CRP level < 6mg/l in the sample.

3.3.2.4 ESR assay ESR was determined by Westergren method.

A. Principle of the method Whole blood (4 volumes) anticoagulated with 0.109 M trisodium citrate (1

volume) is mixed in ESR tube and allowed to stand for exactly 1 hour in a

vertical position. The number of millimeters the RBCs fall during this time

constitutes the ESR value.

B. Method components (Milano com –Italy)

Westergren pipet (calibrated in millimeters), Westergren pipet rack.

C. Procedure 1. Fill the test tube containing 0.2 ml of sodium citrate up to the graduation mark

with blood

2. Mix immediately by gently capsizing three to four times.

3. Insert the pipette through the pierceable stopper, the blood will automatically

rise to zero.

42

The normal values are (0 to 15 mm/hr) for women, (0 to 10mm/hr) for men.

3.4 Statistical methods • The statistical analyses were performed using SPSS version "15". The

sensitivities and specificities were calculated together with the 95% CI by

ROC curve which is generated by plotting sensitivity (y axis) against 1-

specificity (x axis).

• The correlations between numerical data were analyzed by Pearson's

correlation coefficient and Chi-square test of independences was used for

correlation between nominal or order data.

• Comparison of numerical data between groups were made by Mann

Whitney U test and comparison of proportions between groups were

made by Z - test.

• Means, SD, percentage and CI were used where appropriate.

• P-value less than 0.05 was regarded as significant.

43

Chapter IV Results

4.1 Characteristics of the study population The study population comprised 67 patients who showed symptoms of

different forms of arthritis, however, only 34 patients complied with the criteria of

ACR. And the diagnosis was confirmed by a rheumatologist. Others; two

patients had osteoarthritis, one patient had SLE, one patient had

spondyloarthropathay and those patient were categorized as non RA patients.

The rest "29 patients" were categorized as having UA. Controls group included

75 healthy individual.

RA Patients The demographic and clinical characteristics of RA patients are summarized in

Table 4.1. Table 4.1 Characteristics of RA patients (n=34)

Character Mean ± SD or NO (% of patients)

Age

40.6 ±11

Female (%) Male (%)

30 (88.2%) 4 (11.8%)

Disease duration < 1 year 1-5 year > 5 year

6 (17.6%) 16 (47.1%) 12 (35.3%)

Smoking status (current or previous smoker)

3 (9%)

Family history

6 (18%)

Joint swelling

32 (94%)

Extra-articular manifestation

13 (38%)

Radiological manifestation (erosive)

17 (57%)

Drug treatment (DMARD or steroid or anti TNF drugs)

28 (82%)

44

Non RA and UA patients The characteristics of non RA and UA patients are summarized in table 4.2

Table 4.2 Characteristics of non RA and UA patients (n=33)

Character Non RA patients Mean ± SD or

NO (%)

UA patients Mean ± SD or

NO (%) Age 33±13.8 38±11

Female Male

3 (75%) 1 (25%)

22 (76%) 7 (24%)

Smoking status

0 3 (10%)

Family history

1 (25%) 8 (28%)

ESR (mm/h)

44.5±64.4 21.8±19.9

CRP (≥6 mg/I) (<6 mg/I)

1 (25%) 3 (75%)

12 (58%) 17 (42%)

Controls group: The characteristics of controls group are summarized in table 4.3

Table 4.3 Characteristics of controls group (n=75)

Character Healthy individual Mean ± SD or

NO (%) Age 38±15

Female Male

43 (57%) 32 (43%)

Smoking status

5 (7%)

Family history

2 (3%)

ESR (mm/h)

10.7±10.8

CRP (≥6 mg/I) (<6 mg/I)

9 (22%) 66 (88%)

45

Non RA patients, UA patients and Control group was included in the study in

order to assess the specificity of ACCP2A and IgM RF and to demonstrate the

importance of ACCP2A in differentiating the early suspected RA patients (not

yet fulfilling the ACR criteria according to the clinical evaluation) from other

arthritis patients.

4.2 Sensitivity and specificity of ACCP2A and IgM RF for diagnosis of RA

Sensitivity expresses the percentage of RA patients that was positive for

the test, whereas specificity is calculated from the percentage of test-negative

non RA patients, UA patients and healthy control. Both were analysed by ROC

curve at optimal cut off (>5 U/ml) for ACCP2A and at (>6 IU/ml) for IgM RF.

The sensitivities and specificities (with their corresponding 95% CIs) of the

serologic tests are illustrated in Table 4.3. As shown, sensitivity for RA was

higher for IgM RF (85%) than ACCP2A (56%) and specificity was very greater

for ACCP2A (99%) than for IgM RF (69%). While the sensitivity of combination

of these tests (both or one of these tests) was 88% and specificity approached

100%.

Table 4.4 Diagnostic value of ACCP2A and IgM RF for RA

criterion ACCP2A IgM RF Combination of ACCP2A and IgM RF

Sensitivity %

95% CI

56%

85 %

88%

Specificity %

95% CI

99 %

69%

100%

For comparisons of the diagnostic value of each assay, AUC were calculated. It

indicates that for a randomly drawn RA patient and controls, the probability that

the RA patients will have a higher test value than the control; a value of 0.5

46

indicates no discrimination and 1 perfect discrimination. AUC was 0.83 (95% CI,

0.74 – 0.92) for IgM RF and 0.80 (95% CI, 0.70 – 0.90) for ACCP2A with P-

value (sig.) =0.000 for both. Therefore both tests exhibited similar diagnostic

value in Palestinian patients with RA. The ROC curve of ACCP2A and IgM RF

and comparison between them are demonstrated in Figures 4.1, 4.2 and 4.3.

Figure 4.2 ROC curve of IgM RF

AUC = 0.80

1 - Specificity1.00.80.60.40.20.0

Sens

itivi

ty

1.0

0.8

0.6

0.4

0.2

0.0

Cut off > 5

1 - Specificity1.00.80.60.40.20.0

Sens

itivi

ty

1.0

0.8

0.6

0.4

0.2

0.0

Figure 4.2 ROC curve IgM RF

ROC Curve

Figure 4.1 Roc curve of ACCP2A

Cut off > 6

AUC = 0.80

AUC = 0.83

ROC Curve

47

1 - Specificity1.00.80.60.40.20.0

Sens

itivi

ty1.0

0.8

0.6

0.4

0.2

0.0

ROC Curve

Reference LineF

CP2A

urce of the Curve

nal segments are produced by ties.

IgMRAC

So

Diago

Pairwise comparison of ROC curves (ACCP2A ~IgM RF) AUC of ACCP2A= 0.80AUC of IgM RF = 0.83Differences between areas = 0.03

IgM RF curve ACCP2A curve

Figure 4.3 Comparison between ACCP2A and IgM RF ROC curves

4.3 Comparison between the mean levels of ACCP2A and IgM RF in RA and controls plus others groups

We used Kolmogorov-Smirnov test to examine if the distributions for

ACCP2A and IgM RF for the two groups are normally distributed. From the

SPSS result, p-value = 0.000 and as shown in Figures 4.4 and 4.5, most of the

points are not close to the normal line, indicating that the distribution of ACCP2A

and IgM RF for both groups are not normally distributed. Therefore, an

appropriate test is Mann Whitney U test for comparison between groups.

48

Observed Value2001000-100-200

Expe

cted

Nor

mal

Val

ue

200

100

0

-100

Normal Q-Q Plot of IgMRF

Figure 4.4 Test distribution of ACCP2A

Figure 4.5 Test distribution of IgM RF

Observed Value100500-50-100

Expe

cted

Nor

mal

Val

ue

50

25

0

-25

-50

Normal Q-Q Plot of ACCP2A

Since P- value (sig.) = 0.000 which is smaller than the leve

.05), then there exists a significant differences between the

0.000) for each ACCP2A and IgM RF. Therefore based o

mean rank of ACCP2A and IgM RF which are higher in pati

plus others, we concluded that means of ACCP2A and IgM

significantly higher than for controls plus other group

summarized in Table 4.5 and Figure 4.6.

49

l of significance (α =

two groups (sig. =

n this result and the

ents than in controls

RF for patients are

s. The results are

Table 4.5 Comparison between ACCP2A and IgM RF in RA patients (n=34), controls (n=75) and others patients (n=33)

IgM RF (U/ml) Mean ±SD

ACCP2A (U/ml)Mean ±SD

NO Groups

98±76.9 34.5±42.7 34 RA patients

19.9±21 1.1±0.58

4 Non RA patients

48.0±73 2.0±3.8 29 UA patients

9.2±23.7 1.1±0.57 75 Healthy control

107.1 60.3

103.4 61.4

Mean rank RA patients Control +other patients

0.000** 0.000** P value *p< 0.05; **p<0.01; ***p<0.001

healthy disease UA non RA

groups

0.00

20.00

40.00

60.00

80.00

100.00

Mean

IgMRFACCP2A

Figure 4.6 Comparison between ACCP2A and IgM RF in RA patients, controls and others patients.

50

4.4 The concordance between ACCP2A and IgM RF We used Pearson's correlation coefficient to examine the direction and

the strength of the relationship between ACCP2A and IgM RF. As shown in

Table 4.6, the concordance between them was significant correlation. This

means that the two tests have the ability to identify the same patients as positive

or negative.

Table 4.6 The relationship between ACCP2A and IgM RF ACCP2A

RF IgM

Pearson correlation

Sig.(2-tailed)

0.353**

0.000

*p< 0.05; **p<0.01; ***p<0.001

4.5 Prevalence of ACCP2A and IgM RF in RA patients The total number of patients who were positive for ACCP2A was 19

(56%), while those who were positive for IgM RF represented 29 (85%) patients.

IgM RF as the single positive marker was found in 35% and ACCP2A as a

single marker in 6%, this demonstrated the importance of ACCP2A in diagnosis

of sero negative RA. And the positive patients for both ACCP2A and IgM RF

were 50% Table 4.7 and Figure 4.7.

Table 4.7 Cross tabulation of ACCP2A and IgM RF in RA patients (n=34) ACCP2A (+)

NO (%) ACCP2A (-)

NO (%) Total

NO (%) IgM RF (+) 17 (50) 12 (35)

29 (85)

IgM RF (-) 2 (6 )

3 (9) 5 (15)

Total 19 (56)

15 (44)

34 (100)

51

ACCP2A(+),RF(+) (n=17)ACCP2A(-),RF(+) (n=12)ACCP2A(-),RF(-) (n=3)ACCP2A(+),RF(-) (n=2)

50%

35%

9%

6%

Figure 4.7 Frequency of ACCP2A and IgM RF in RA patients

4.6 Prevalence of ACCP2A and IgM RF in controls group and others patients

There weren't positive numbers of ACCP2A in non RA patients while IgM

RF gives 2 (50%) positive numbers in them. In UA patients, only 1 (3.4%)

positive for ACCP2A and 16 (48%) for IgM RF and no positive numbers of

ACCP2A in healthy controls, but IgM RF gives 17 (23%) positive numbers,

Table 4.8.

Table 4.8 Prevalence of ACCP2A and IgM RF in controls group and others patients

Group Total positive NO of ACCP2A

Total positive NO of IgM RF

Non RA patients 0 2 (50%)

UA patients 1 (3.4%) 16 (48%)

Control 0 17 (23%)

52

4.7 A correlation of ACCP2A and IgM RF with inflammatory parameters (CRP and ESR) and disease activity parameters (radiological manifestation and extra-articular manifestation) The correlation of numerical data such as ESR variables with ACCP2A

and IgM RF were analysed by Pearson's correlation coefficient and the

correlation of ordinal data and nominal data such as CRP, extraarticular and

radiological manifestations with ACCP2A and IgM RF are analysed by Chi

square test.

Table 4.9 shows that a significant correlation was found between ACCP2A and

radiological manifestations, extra-articular manifestations and CRP and no

correlation existed between ACCP2A and ESR. In addition there is significant

correlation between IgM RF and radiological manifestation, extra-articular

manifestation, CRP and ESR.

Table 4.9 Correlation between Abs assay and inflammatory and disease activity parameters

Criterion

ACCP2A Pearson correlation

Sig (2- tailed)

IgM RF Pearson correlation

Sig (2- tailed) Radiological manifestation

Pearson chi square Sig. (2-tailed)

22.045

0.000**

17.260

0.001** Extra-articular manifestation

Pearson chi square Sig. (2-tailed)

15.068

0.001**

16.169

0.001** CRP

Pearson chi correlation Sig. (2- tailed)

7.468

0.024*

36.679

0.000** ESR

Pearson correlation Sig. (2- tailed)

0.005 0.952

0.190

0.023* *p< 0.05; **p<0.01; ***p<0.001

53

4.8 Comparison between ACCP2A (+) and ACCP2A (-) In total 34 patients; 19 of them were positive for ACCP2A and 15 patients

were negative for ACCP2A. Summary of different variables in relation to

ACCP2A results are presented in Table 4.10. Differences in means between

groups were analyzed with the Mann-Whitney test and proportions were

compared using the Z- test. From table 4.10, the disease duration between two

groups isn’t significant; (<1year p= 0.05, 1-5year p= 0.12, >5year p= 0.2). There

is also insignificant differences between the two groups in relation to early

symptoms (pain p= 0.28 , swelling p= 0.27 , function loss p= 0.42, redness p=

0.27). However, early symptoms started in the small joints showed significant

difference between groups (p=0.01), ACCP2A positive had higher percentage of

patients with initial joint symptoms in small joints than ACCP2A negative (42% v

20% ). But there is no significant difference in localization of initial joint

symptoms in large joint or both (p=0.2, p=0.35). In conclusion there are no

considerable differences in the early symptoms of disease between ACCP2A

positive and ACCP2A negative RA patients. There is a significant difference

between ACCP2A positive and ACCP2A negative in extra-articular

manifestations (p=0.006), since ACCP2A positive patients had higher

percentage of extra-articular manifestations than ACCP2A negative patients

(47% v 20%). On the other hand, there was no significant difference between

both groups in radiological manifestations (0.12). Also there was no significant

difference in the proportion of patients who received methotrexate, antimalarial

or combination of DMARD treatments (p = 0.05, 0.5 and 0.5). However, a

significant difference was found in the proportion of patients who received

steroidal and other drugs such as (Enbrel, anaflam, penicillamine and poverty

medications), since ACCP2A positive had higher percentage of treatment with

these drugs than ACCP2A negative (26% v 13%) and (37% v 7%). In addition,

there was no significat difference in the proportion of CRP positive and negative

between the two ACCP2A groups (p=0.31 for CRP ≥6 and p=0.24 for CRP <6)

54

and in the mean levels of ESR (P= 0.12). In conclusion: the two groups are

relatively similar in the phenotype of RA.

Table 4.10 Comparison of patients who are ACCP2A (+) and who are ACCP2A (-) in

relation to different variables variables

ACCP2A Positive patients (n = 19 )

ACCP2A Negative patients (n = 15)

P value

Disease duration < 1 year 1-5 year >5 year

4 (21)

10 (53) 5 (26)

2 (13) 6 (40) 7 (47)

0.05 0.12 0.20

Initial symptoms Pain Swelling Function loss Redness

19 (100)

18 (95) 12 (63) 14 (74)

15 (100)

14 (93) 13 (87) 11 (73)

0.28 0.27 0.42 0.27

Localization of initial joint symptoms Small joints Large joints Both

8 (42) 4 (21) 7 (37)

3 (20) 6 (40) 6 (40)

0.01* 0.2 0.35

Extra-articular involvement

9 (47) 3 (20) 0.006**

Erosive 10 (53) 6 (40) 0.12 ESR (mm/h) 19 ±10 31 ±32 0.12 CRP (≥6 mg/l) (<6 mg/l)

11(58) 8 (42)

9 (60) 6 (40)

0.31 0.24

Drug treatment [DMARD] MTX Antimalarial drugs Avara Combination [Steroidal drug] [Other drug]

4 (21) 4 (21)

1 (5) 3 (16) 5 (26) 7 (37)

2 (13) 4 (27)

3 (20) 2 (13)

1 (7)

0.05 0.5

0.5

0.02* 0.0001***

Values are mean (SD) or n (%). MTX; methotrexate, antimalarial drugs; chloroquine phosphate or hydroxychloroquine and other, for example, Enbrel, anaflam, penicillamine and poverty medications. *p<0.05 ; **p<0.01; ***p<0.001

55

4.9 Relation of ACCP2A and IgM RF with personal characters of RA patients (age, gender, family history and smoking)

We studied the relationship of ACCP2A and IgM RF with numerical data

such as age variable by Pearson's correlation coefficient and with nominal data

such as gender, family history and smoking by Chi square test of

independences. As shown in table 4.11, there was no significant relationship

between both ACCP2A and IgM RF with age. On the other hand, there was a

significant relationship between IgM RF and gender. However, this relation

wasn't significant for ACCP2A. In addition, there was no significant relationship

between ACCP2A and IgM RF with family history or smoking.

Table 4.11 Relation of ACCP2A and IgM RF with personal characters of RA patients

Risk factors

ACCP2A IgM RF

Age Pearson correlation

Sig. (2- tailed)

0.076 0.367

0.031 0.718

Gender Pearson chi square

Sig. (2- tailed)

3.739 0.154

11.974

0.007* Family history

Pearson chi square Sig. (2- tailed)

3.951 0.139

3.508 0.320

Smoking Pearson chi square

Sig. (2- tailed)

2.460 0.292

4.415 0.220

56

Chapter V Discussion

RA is associated with significant morbidity and mortality. There is likely to

be a ‘window of opportunity’ early in the disease when disease-modifying

treatment can significantly alter the disease course and prevent or modify the

development of erosions which can eventually lead to joint failure, and even

mortality.

Best practice is to identify and treat the disease early. RA remains a clinical

diagnosis, especially very early in the course of the disease when radiological

change specific to RA or erosions may not yet be evident. Early identification

and treatment might be more accurately realised. This is particularly exciting at a

time when our approach to treatment has been revolutionized by the advent of

the concept of tight RA disease control and with both combination of

conventional DMARDs and with biologic therapies [134].

RF assay has known to have suboptimal sensitivity and specificity for the

diagnosis of RA. Therefore, easier, more convenient, higher specific serological

methods have been required for the diagnosis of RA [135]. Now the diagnostic

properties of ACCP2A in the specific diagnosis of RA may outplay other

available antibody tests, especially IgM-RF [136]. This study aimed at

comparing ACCP2A assay with IgM RF test.

57

5.1 Diagnostic performance of ACCP2A 5.1.1 Sensitivity of ACCP2A and IgM RF

In the present study, the sensitivity of IgM RF was moderately high 85%

and it is in agreement with most studies which resulted in sensitivity about 80%

[137,138]. ACCP2A showed good sensitivity (56%), however there is

considerable variation in diagnostic sensitivity of ACCP2A among studies,

ranging from 41%–80% [139-142]. The reasons of the wide range of sensitivity of reported results of ACCP2A may

include:

1. The cut-off value used to define a positive result varied significantly in articles,

even if provided by the same manufacturer. This difference in terms of sensitivity

in the same serum samples between manufacturers was observed by Garcia-

Berrocal [143]. Indeed, cut-off level for positivity was either determined from

OD450 (≥0.11 or 0.3) or expressed in units (determined from manufactures or

from a ROC curve) [144]. So some standardization would appear desirable, with

determination for each manufacturer for the optimum cut-off value.

2. As ACCP2A measurements weren't followed-up, so we cannot exclude the

possibility that some RA patients that were ACCP2A negative at inclusion have

become ACCP2A positive at a later stage in the disease and this was reported

by Schellekenes and his colleagues [145]. So serial determination of ACCP2A

from one time to another is important, especially for those whom results are in

borderline (in our study 5 patients have >4 U/ml of ACCP2A concentration).

3. The characteristics of patients were evaluated, e.g. in our study some patients

(who are fulfilling ACR and suspected RA) after follows up clinically by

rheumatologist after 10 months from blood sampling, they were diagnosed as

having other disease of autoimmunity such as SLE. This reflects the relatively

low specificity of ACR criteria in diagnosis of RA and emphasizes the

importance of ACCP2A in differentiating RA from other types of arthritis.

4. Ethnicity of patients. ACCP2A was studied in large number of ethnic groups

but its pathogenesis until now isn’t known. So some triggers that are related to

58

ethnicity may have an effect on the production of ACCP2A, in this respect, the

role of specific genes shouldn't be ignored. 5. One could ask whether ACCP2A negative patients do not have Abs to

citrullinated epitopes. Because RA is a complex disease [146], so reaching

100% sensitivity will be impossible. However, the results of several experiments

indicated that the Ab response to citrullinated epitopes is strongly polyclonal

[147]. Many researchers nowadays are concentrating their researches on how

they could improve the sensitivity of ACCP2A. One of these studies suggested

that the use of new ACCP3A assay which contains additional number of

citrullinated epitopes can enhance the clinical sensitivity for diagnosis of RA

[148]. Another study made by Hueber and his colleagues that identified

peripheral blood Ab and cytokine profiles that characterise clinically relevant

subgroups of patients with early RA using arthritis Ag microarrays and a

multiplex cytokine assay. They found increased blood levels of proinflammatory

cytokines which are associated with Ab targeting of citrullinated Ags in patients

with early RA. So they expected that proteomic analyses will become a mainstay

in the evaluation of patients with RA and other autoimmune diseases for

assessing prognosis, guiding treatment and monitoring response to treatment

[149].

5.1.2 Specificity of ACCP2A and IgM RF The specificity of the ACCP2A test (99%) was significantly higher than

that obtained for the IgM RF test (69%). The specificity of IgM RF is in

concordance with other studies which is about 70% [150,151]. There is also

consensus of the different studies about the high specificity of ACCP2A ranging

between 95% and 100% [152,153]. The specificity is the most valuable aspect

of this assay, so much so that it may be proposed as the most important

examination in the diagnosis of RA.

59

5.1.3 Sensitivity and specificity of combination (both or one) of ACCP2A and IgM RF

Our study also showed that the combination of ACCP2A with IgM-RF

could increase the sensitivity (to 88%) and specificity (to 100%). The

complementarity of RF to ACCP2A is controversial. Some reports suggested that

RF and ACCP2A should be combined to reach optimum diagnostic properties,

whereas others found only little additional diagnostic value when combining RF

and ACCP2A. The discrepancy could be explained by the fact that in the

literature stratification for specificity was not performed, and therefore, the results

of different studies are not comparable. Furthermore international discussion is

warranted to answer the question of whether ACCP2A should replace or

complement RF in the future, especially as RF constitutes one of the revised

ACR criteria for RA [154]. However to answer such question we believe that

many studies comprising large different populations should be made and an

agreement must be faithful in the represented sample selection (patients and

control groups), kits (from the same manufactures) and the used method to

calculate the sensitivity and specificity of combination of ACCP2A and IgM RF.

5.1.4 Comparison the diagnostic accuracy between ACCP2A and IgM RF

The diagnostic accuracy between ACCP2A and IgM RF was compared,

and the AUC of ACCP2A and IgM RF was 0.80 and 0.83, respectively.

Diagnostic accuracy between them was not different; this result is similar to

other studies [155,156]. But differ from other result which indicates that

ACCP2A showed a better diagnostic performance than IgM RF [157-159]. The

difference between studies in this issue is due to different cutoff levels, different

patient populations and small sample size of our study. However, the difference

in mean levels of ACCP2A and IgM RF between RA patients and others groups

was significant (p=0.000) and this is another point which emphasizes that

ACCP2A and IgM RF are having similar diagnostic accuracy.

60

5.2 Diagnosis of sero-negative RA by ACCP2A The diagnosis of sero-negative RA is a more challenging diagnosis, and

more likely to include misclassified reactive, psoriatic, viral and crystalline

arthritides [160]. Our study shows that; although the two Abs were significantly

correlated (Pearson correlation, 0.353**; p=0.000), ACCP2A was present alone

in 6% of IgM-RF negative sera of RA patients. This result confirms the

hypothesis that "ACCP2A and RF are distinct Ab systems yielding different

information" [161]. And also indicate that ACCP2A estimations may be useful in

the disease diagnosis of RA patients who are RF-negative or where the

diagnosis may be uncertain and this added diagnostic and prognostic value for

ACCP2A.

5.3 ACCP2A is predictive for the outcome of early RA Next to being sensitive and specific, good markers of disease should be

detectable as early in the disease process as possible. In this study 3.4% of UA

patients, could not fulfill ACR criteria and were classified as UA, showed

positivity toward ACCP2A. Many studies demonstrated that, the presence of

ACCP2A might be a prognostic factor for future onset of RA [162-164]. These

Abs appear in the serum many years before the onset of clinical disease [165]. This suggests that the initial trigger for the development of RA may occur long

before the appearance of symptoms. Monitoring ACCP2A activity in individuals

that may have an increased risk for the development of RA, e.g. based upon

genetic factors, may eventually allow earlier treatment of ACCP2A positive

individuals in which the Ab titers are increasing. As a consequence, the lag time

between first visit to the rheumatology clinic and start of therapeutic intervention

may be importantly reduced and joint erosion may already be inhibited at the

very early stages Figure 5.1 [166].

61

Figure 5.1 Presence of ACCP2A precedes the clinical onset of RA by years [166].

ACCP2A appears early in the disease course and has a high specificity for RA.

Therefore it might be a valuable additional predictive tool in the management of

this disease.

5.4 Use of ACCP2A to differentiate RA from other similar diseases

Many rheumatic or immune diseases can present the clinical symptoms

of polyarticular, symmetrical arthritis and positive RF, which mimic RA or fulfill

the diagnosis of RA. These consist of SLE, PSA with polyarticular involvement,

Human Immunodeficiency Virus - related arthropathy, PMR, and even UA or PR

[167]. In our study; all non RA patients (SLE, osteoarthritis and

spondyloarthropathy patients) were negative for ACCP2A test but 50% of those

patients were positive for IgM RF. This means that ACCP2A may be useful in

distinguishing RA from other similar diseases and RF is unhelpful in these

conditions. This finding is in consensus with other studies [168-172]. These data

and the absence of ACCP2A in healthy individuals and in other rheumatic

diseases confirm their high propensity to be associated with RA.

62

5.5 Prognostic ability of ACCP2A: In our study both of ACCP2A and IgM RF were found to have significant

correlation with radiological manifestation (p=0.000). This is in accordance with

other results that support the presence of both ACCP2A and IgM RF as the best

predictors of erosive or progressive disease [173]. Other studies even

demonstrated that ACCP2A was appearing stronger in correlation with

radiological manifestations than IgM RF [174,175]. For this reason ACCP2A

became an important part of a newly proposed model for diagnostic criteria of

early arthritis and strongly predicted persistent and erosive disease as

previously described in literature section. However, one study claimed that RF

was the main factor predicting radiological progression [176].

The relation of both ACCP2A and IgM RF with extra-articular manifestations was

also significant (P=0.000). This finding is in accordance with other authors

[177,178]. On the other hand, Korkmaz and his colleagues didn't find a

relationship between ACCP2A and IgM RF and extra-articular manifestations

[179]. In this context, our findings revealed that the presence of ACCP2A and

IgM RF act as indicators of disease severity in RA, and the presence of both

suggest a role for these Abs in the pathogenesis of radiological and extra-

articular manifestation.

The correlations of ACCP2A and IgM RF with CRP and ESR were studied. CRP

and ESR are, along with the number of swollen and painful joints, one of the

major criteria for the “clinical” disease activity score and the ACR scores [180]. In our study there was significant correlations between ACCP2A and IgM RF

with CRP (p=0.005 and p=0.000). This is in accordance with some reports

[181,182]. But, it remains unclear whether increased inflammation, measured by

CRP, occurs before, after or simultaneously with the development of Abs (IgM

RF and/or ACCP2A) in the preclinical phase of RA [183]. These results are

opposing Berglin who didn’t found any correlation between ACCP2A and IgM

63

RF and CRP [184]. The significant correlation of ESR was found only with IgM

RF (p=0.023) and this in consensus with Aldifran [182], and in conflict with

Stropuvienë [185]. And the correlation of ESR with ACCP2A wasn't significant

(p=0.952), this is in consensus with Shovman [186].

It is interesting to note that IgM RF has more correlation with CRP than ACCP2A

(36.679 v 7.468). In addition it has significant correlation with ESR rather than

ACCP2A (0.023 v 0.952). This will support the data in recent published research

which demonstrated that the levels of ACCP2A don't change depending on anti-

TNF treatment, in contrast to RF, CRP and ESR. It has been concluded that IgM

RF and ACCP2A are two different systems of Abs reacting differently to the

treatment prescribed [187]. However the patients included in this study were

under the different regimes of treatment (DMARD, steroids, anti-inflammatory

drugs). So further studies should be made to demonstrate the reactions of

ACCP2A with each drug.

The reasons standing behind results discrepancy may include:

1. Some studies were cross-sectional in this issue and therefore they are

subjected to possible selection bias. Prospective studies would probably

have produced more information than a cross-sectional study of patients

with definite RA. However, in prospective studies, it is necessary to

assume that treatments have not biased the results. They are therefore

subject to ethical problems or to the possible loss of sensitivity as new

and more effective treatments, adjustable to disease severity, become

available. 2. Methodological differences in the assays used. 3. Different cut off values chosen for ACCP2A and IgM RF. 4. Differences in patient selection (with current, moderate or severe disease

and different life style). 5. Small sample size. 6. Treatment.

64

7. Disease duration.

5.6 Why ACCP2A RA patients have negative results? In this study the phenotype of RA patients with or without ACCP2A did

not differ exactly at clinical presentation. We observed neither a significant

difference in the reported early symptoms (pain; p= 0.28, swelling; p= 0.27,

function loss; p= 0.42, redness; p= 0.27) nor in localization of initial joint

symptoms in large joints (p=0.2) or in both joints (p=0.35) except in small joints,

since there were significant difference between two groups (p=0.01).

Although, there wasn't significant differences between ACCP2A positive and

ACCP2A negative RA patients in erosive manifestations (p=0.12). The

significant difference appears between ACCP2A positive and ACCP2A negative

in extra-articular manifestations (p=0.006). However, no significant differences

were observed in inflammatory parameters CRP (≥6; p=0.31, <6; p=0.24) and

ESR (p=0.12). This implies that although different associations with known risk

factors are reported for ACCP2A positive and ACCP2A negative RA patients,

the presence or absence of ACCP2A is not associated with a distinguishable

clinical phenotype at presentation of disease.

Pathophysiology may have implications; It was recently observed that the

prominent genetic risk factor HLA class II alleles only associate with

susceptibility to RA in the presence of ACCP2A but not with RA in the absence

of these Abs [188]. It has been shown that the conversion of arginine to citrulline

in HLA-DRB1*0401 transgenic mice has been demonstrated to significantly

increase activation of CD4+ T cells. In a study of patients with early RA, a

significant association between ACCP2A and expression of B1*0401/0101 was

reported. This finding suggests that individuals carrying the SE genes may have

more sustained T- and B-cell responses to citrullinated Ags than noncarriers.

Taken together, these data suggest that in individuals carrying one or two SE

65

genes, a specific T-cell-dependent immune response to citrullinated peptides

may contribute to the occurrence of RA [189]. However, in the present study, no

attempt has been made to investigate the relationship of RA with these factors

due to the lack of facilities.

Although differences in risk factors presume different pathophysiological

pathways for ACCP2A positive RA and ACCP2A negative RA, the initial

phenotypical presentation of both patient groups is relatively similar and is

characterized by a symmetric polyarthritis of the same large joints or both joints

(small and large). This leads to a pathophysiological model in which one or more

triggers lead to arthritis in similar joints in ACCP2A positive patients and

ACCP2A negative patients. Ags are subsequently citrullinated during

inflammation; in the presence of ACCP2A the inflammation is aggravated,

resulting in more severe radiological destruction [190]. RA is variable course, so

absence of ACCP2A in RA patients may indicate to self limiting of this group

from disease in future. However further studies are needed to add insight into

the pathogenic role of circulating ACCP2A in ACCP2A positive RA and to

unravel the risk factors associated with ACCP2A negative RA.

Furthermore, treatment with DMARDs (MTX; p=0.05, Antimalarial drugs; p=0.5

or Combination; p= 0.5) wasn't significantly different between the two groups but

receiving steroidal drug and other drugs such as ( Enbrel, anaflam, peniciiamine

and proverty medications showed significant difference (p=0.02, 0.0008). Since

ACCP2A positive patients are receiving more steroidal (26% v 13%) and other

drugs (37% v 7%) than ACCP2A negative patients. This is reflecting the more

aggressive disease in this group of ACCP2A positive patients than in the

ACCP2A negative group. However this did not prevent the development of more

radiological destruction and more extra-articular in the RA patients with

ACCP2A. This finding is in concordance with Kastbom [191]. The stability of

ACCP2A status and the failure of efficient treatment to eliminate these Abs from

66

serum strengthen the notion that ACCP2A positive RA may differ from ACCP2A

negative RA in several important aspects, and that these two subgroups thus

should be evaluated separately in future studies of both etiology and treatment

[192].

5.7 Correlation of ACCP2A and IgM RF with personal characters of RA patients

Most studies have shown that there was no correlation between ACCP2A

with age or gender [193,194]. This has also been confirmed in our study. There

was also no correlation between IgM RF and age. Similar results were obtained [193,194]. However, significant correlation was found between IgM RF and

gender (p=0.007) and this is in consensus with Masi [195]. The strong

correlation of IgM RF with gender is an indication that gender could act as a risk

factor for RA and the weak correlation of ACCP2A with gender may be due to

the moderate sensitivity of ACCP2A in our study. In addition, variations in

hormonal levels between male and female may influence the results.

Various studies have shown that smoking is a predictor of the development of

RA [196,197]. In our study there was no correlation of ACCP2A and IgM RF with

smoking. This may be due to the fact that most of the patients in this study were

female and smoking habit was more among male patients than females. In

addition epidemiologic studies have revealed that the risk of cigarette smoking

for seropositive RA is higher in men (odd,s ratio: 4.77 ) than in women (odd,s

ratio: 1.14) [198].

Our study showed that there is no correlation of ACCP2A and IgM RF with

family history. This is in concordance with van Gaalen who found that other

groups at risk of developing RA, such as family members of RA patients or even

the general public, are less likely to benefit from ACCP2A testing due to the low

prevalence of RA 40% [199]. In addation, Svendsen showed that Genes are of

67

minor importance in the development of RA [200]. This point needs further

research on all family members to reach concrete conclusion. Finally, RA is still

of unknown etiology. Environmental and genetic risk factors have been

identified, but no single risk factor has emerged as necessary or sufficient to

cause the disease.

5.8 Adopting model for lab diagnosis of RA in Gaza Strip Because ACCP2A has excellent specificity, we think that it might be more

advisable to determine ACCP2A first and then RF in ACCP2A negative patients,

or ideally, both Abs in parallel to increase the likelihood of identifying patients

with RA who do not have RF but who have other RA markers. The combination

of a positive ACCP2A and IgM RF result is highly specific for RA 100% and is

highly sensitive for RA 88%.

RA Diagnostic panel

(IgM RF and IgG ACCP2A)

ACCP2A + IgM RF +

ACCP2A + IgM RF -

ACCP2A - IgM RF +

ACCP2A – IgM RF -

Consistent with RA; Predictive of

progressive joint destruction

Frequently present in early RA; Predictive of

progressive joint destruction

More suggestive of other rheumatic

disease or infection, but consistent with RA

RA unlikely; observed in only 10% to 20% of

patients with RA

Figure 5.2 Is it RA? [201]

68

Chapter VI Conclusions and Recommendations 6.1 Conclusions 1. ACCP2A assay is a very valuable tool for the diagnosis of RA. This ELISA

test avoids the problems of RF tests regarding to low specificity and false

negative in RA patients. Moderate sensitivity of ACCP2A does not allow using

as a screening test, but because of high specificity of ACCP2A, especially when

high Ab titers are present, it may become one of the most useful serologic tests

for the diagnosis of RA in Gaza Strip.

2. The use of ACCP2A and RF tests combined is considered to be the ‘gold

standard’ in the detection of RA, because the combination of them provides

nearly 100% specificity and 88% sensitivity and thus could be helpful in the

differential diagnosis of RA and other rheumatic diseases. In addition, this test

may be very influential for the rheumatologists to chose the best therapeutic

strategy in patients with recent-onset arthritis.

3. The presence of ACCP2A in patients with UA is indicative of the probability

that they will develop RA. And this reflects the importance of ACCP2A in

diagnosis of early RA.

4. Both of ACCP2A and IgM RF are associated with radiological manifestations,

extra articular manifestations and inflammatory parameters such as CRP. This

suggests a role for these Abs in disease activity. They could be of use as

markers for severe disease outcome.

5. The clinical presentation of RA patients with or without ACCP2A is relatively

similar. However, patients with ACCP2A develop a more severe disease course

compared with RA patients without these Abs. This means that the two Abs

69

have separate risk factors and which has been confirmed in other recent

studies. This may explain the moderate sensitivity of ACCP2A in our study. 6.2 Recommendations 1. The ELISA ACCP2A assay provides a reliable, cost-effective possibility to test

ACCP2A in Gaza Strip laboratories.

2. Future developments which should be discussed include that the presence of

ACCP2A could be added to the ACR criteria for RA.

3. Investigation of the correlation of risk factors of RA especially HLA RR β1

genes with ACCP2A to identify the patients who are at high relative risk for

future development of RA, is recommended.

4. Two subgroups (ACCP2A positive and ACCP2A negative) should be

evaluated separately in future studies for both aetiology and treatment.

5. Research that looks forward for the improvement of sensitivity of ACCP2A by

either generation of ACCP3A or microarray, should be followed up.

6. Study whether monitoring the level of ACCP2A Abs will be useful as a marker

of disease control in RA patients in Gaza Strip.

7. As there is scarcity of studies about rheumatism diseases in this area, so we

suggest conducting more research in this field.

8. Awareness programs should be launched especially for persons who are

liable to get RA.

70

Reference______________________________________________ [1] Abreu I, Laroche P, Bastos A, et al., 2005 - Multiplexed Immunoassay for

Detection of Rheumatoid Factors by FIDIS Technology. Ann. N.Y. Acad. Sci,

1050:357–363.

[2] Jawaheer D, Lum R, Gregersen P and Criswell L., 2006 - Influence of

Male Sex on Disease Phenotype in Familial Rheumatoid Arthritis. Arthritis

Rheum, 54(10):3087-94.

[3] Ceccato F, Roverano S, Barrionuevo A, Rillo O and Paira S., 2006 - The

role of anticyclic citrullinated peptide antibodies in the differential diagnosis of

elderly-onset rheumatoid arthritis and polymyalgia rheumatica. Clin Rheumatol,

25(6):854-7.

[4] Alamanos Y, Voulgari P and Drosos A., 2005 - Rheumatoid Arthritis in

Southern Europe: Epidemiological, Clinical, Radiological and Genetic

Considerations. CRR, 1:33-36.

[5] Edwards C and Cooper C., 2005 - Early environmental factors and

rheumatoid arthritis. Clin Exp Immunol, 143: 1-5.

[6] Gossec L, Dougados M, Goupille P, et al., 2004 - Prognostic factors for

remission in early rheumatoid arthritis: a multiparameter prospective study. Ann

Rheum Dis, 63(6):675-80.

[7] van Venrooij W, Hazes J and Visser H., 2002 - Anticitrullinated

protein/peptide antibody and its role in the diagnosis and prognosis of early

rheumatoid arthritis. Neth J Med, 60(10):383-8.

71

[8] Arnett F, Edworthy S, Bloch D, et al., 1988 – The American Rheumatism

Association 1987 revised criteria for the classification of rheumatoid arthritis.

Arthritis Rheum, 31(3):315–324.

[9] van Riel P., 2002 - Are prognostic factors in rheumatoid arthritis of any use

in daily clinical practice?. J Med, 60(8):381-382.

[10] Weyand C and Goronzy J., 2006 - Pathomechanisms in rheumatoid

arthritis — time for a string theory?. J Clin Invest, 116(4):869-71.

[11] Spiritus T, Verschueren P, Westhovens R and Bossuyt X., 2004 -

Diagnostic characteristics of a gelatin based Waaler-Rose assay (Serodia-RA)

for the detection of rheumatoid factor. Ann Rheum Dis, 63(9):1169-71.

[12] Mikuls T, O'Dell J, Stoner J, Parrish L, Arend W, Norris J and Holers V

., 2004 - Association of Rheumatoid Arthritis Treatment Response and Disease

Duration With Declines in Serum Levels of IgM Rheumatoid Factor and Anti–

Cyclic Citrullinated Peptide Antibody. Arthritis Rheum, 50(12):3776-82.

[13] Kuhn K, Kulik L, Tomooka B, Braschler K, Arend W, Robinson W and

Holers V., 2006 - Antibodies against citrullinated proteins enhance tissue injury

in experimental autoimmune arthritis. J Clin Invest, 116(4):961-73.

[14] van Gaalen F, Ioan-Facsinay A, Huizinga T and Toes R., 2005 - The

Devil in the Details: The Emerging Role of Anticitrulline Autoimmunity in

Rheumatoid Arthritis. J Immunol, 175(9):5575-80.

[15] Schellekens G, de Jong B, van den Hoogen F, van de Putte L, and van Venrooij W., 1998 - Citrulline is an Essential Constituent of Antigenic

72

Determinants Recognized by Rheumatoid Arthritis–specific Autoantibodies. J.

Clin. Invest, 101(1):273 - 281. [16] Takizawa Y, Suzuki A, Sawada T, Ohsaka M, Inoue T, Yamada R and Yamamoto K., 2006 - Citrullinated fibrinogen detected as a soluble citrullinated

autoantigen in rheumatoid arthritis synovial fluids. Ann Rheum Dis, 65:1013 -

1020.

Allan S. Wiik [17] wiik A, van Venrooij W., 2003 - The Use of Anti-cyclic Citrullinated Peptide

(anti-CCP) Antibodies in RA. American College of Rheumatology.

(http:// www.rheumatology.org (last accessed in 25/10/2007)).

[18] Bas S, Genevay S, Meyer O and Gabay C., 2003 - Anti-cyclic citrullinated

peptide antibodies, IgM and IgA rheumatoid factors in the diagnosis and

prognosis of rheumatoid arthritis. Rheumatology (Oxford), 42(5):677-80.

[19] Turesson C and Matteson E., 2006 - Genetics of Rheumatoid Arthritis.

Mayo Clin Proc, 81(1):94-101.

[20] Mimori T., 2005 - Clinical Significance of Anti-CCP Antibodies in

Rheumatoid Arthritis. Intern Med, 44(11):1122-6.

[21] http://en.wikipedia.org/wiki/Rheumatoid_arthritis. (Last accessed in

25/10/2007)

[22] Ewa Berglin., 2006 - Predictors of Disease Onset and Progression in Early

Rheumatoid Arthritis. (Dissertions). UMGA University medical Dissertions,

Sweden.

73

[23] Minnock P, FitzGerald O and Bresnihan B., 2003 - Women with

established rheumatoid arthritis perceive pain as the predominant impairment of

health status. Rheumatology (Oxford), 42(8):995-1000.

[24] http://www.nih.gov/about/researchresultsforthepublic/arthritis. (last

accessed in 25/10/2007)

[25] Avcin T, Cimaz R, Falcini F, et al., 2002 - Prevalence and clinical

significance of anti-cyclic citrullinated peptide antibodies in juvenile idiopathic

arthritis. Ann Rheum Dis, 61(7):608-11.

[26] Dejaco C, Klotz W, Larcher H, Duftner C, Schirmer M and Herold M., 2006 - Diagnostic value of antibodies against a modified citrullinated vimentin in

rheumatoid arthritis. Arthritis Res Ther, 8(4):R119.

[27] Alamanosa Y and Drosos A., 2005 - Epidemiology of adult rheumatoid

arthritis. Autoimmun Rev, 4:130-136.

[28] Rindfleisch J and Muller D., 2005 - Diagnosis and Management of

Rheumatoid Arthritis. Am Fam Physician, 72:1037-47, 1049-50.

[29] Jawaheer D, Lum R, Amos C, Gregersen P and Criswell L., 2004 -

Clustering of Disease Features Within 512 Multicase Rheumatoid Arthritis

Families. Arthritis Rheum, 50(3):736-41.

[30] Pierer M, Kaltenhäuser S, Arnold S, Wahle M, Baerwald C, Häntzschel H, Wagner U., 2006 - Association of PTPN22 1858 single-nucleotide

polymorphism with rheumatoid arthritis in a German cohort: higher frequency of

the risk allele in male compared to female patients. Arthritis Res Ther, 8(3):R75.

74

[31] Hinks A, Worthington J and Thomson W., 2006 - The association of

PTPN22 with rheumatoid arthritis and juvenile idiopathic arthritis. Rheumatology

(Oxford), 45(4):365-8.

[32] Ollier W.E, Harrison B and Symmons D., 2001 - What is the natural

history of rheumatoid arthritis?. Best Pract Res Clin Rheumatol, 15(1):27-48.

[33] Linn-Rasker S, van der Helm-van Mil A, van Gaalen F, et al., 2006 -

Smoking is a risk factor for anti-CCP antibodies only in rheumatoid arthritis

patients who carry HLA-DRB1 shared epitope alleles. Ann Rheum Dis,

65(3):366-71.

[34] Turesson C, Schaid D, Weyand C, et al., 2005 - The impact of HLA-

DRB1 genes on extra-articular disease manifestations in rheumatoid arthritis.

Arthritis Res Ther, 7(6):R1386-93.

[35] Gorman J, Lum R, Chen J, Suarez-Almazor M, Thomson G and Criswell L., 2004 - Impact of shared epitope genotype and ethnicity on erosive

disease: a meta-analysis of 3,240 rheumatoid arthritis patients. Arthritis Rheum,

50(2):400-12.

[36] Goëb V, Dieudé P, Vittecoq O, et al., 2005 - Association between the

TNFRII 196R allele and diagnosis of rheumatoid arthritis. Arthritis Res Ther,

7(5):R1056-62.

[37] Reveille J., 2006 - The genetic basis of autoantibody production.

Autoimmun Rev, 5(6):389-98.

[38] Johansson M, Arlestig L, Hallmans G and Rantapää-Dahlqvist S., 2006 - PTPN22 polymorphism and anti-cyclic citrullinated peptide antibodies in

75

combination strongly predicts future onset of rheumatoid arthritis and has a

specificity of 100% for the disease. Arthritis Res Ther, 8(1):R19.

[39] Plenge R, Padyukov L, Remmers E, et al., 2005 - Replication of putative

candidate-gene associations with rheumatoid arthritis in >4,000 samples from

North America and Sweden: association of susceptibility with PTPN22, CTLA4,

and PADI4. Am J Hum Genet, 77(6):1044-60.

[40] Iwamoto T, Ikari K, Nakamura T, et al., 2006 - Association between

PADI4 and rheumatoid arthritis: a meta-analysis. Rheumatology (Oxford),

45(7):804-7.

[41] van Aken J, van Bilsen J, Allaart C, Huizinga T and Breedveld F., 2003

- The Leiden Early Arthritis Clinic. Clin Exp Rheumatol, 21(5 Suppl 31):S100-5.

[42] Turesson C, Weyand C and Matteson E., 2004 - Genetics of rheumatoid

arthritis: Is there a pattern predicting extraarticular manifestations?. Arthritis

Rheum, 51(5):853-63.

[43] Carty S, Snowden N and Silman A., 2004 - Should infection still be

considered as the most likely triggering factor for rheumatoid arthritis?. Ann

Rheum Dis, 63:46-49.

[44] Riikka Nissinen., 2003 - Immunological features of chronic active rheumatoid arthritis. (dissertation). University of Helsinki, Finland.

[45] Klareskog L, Alfredsson L, Rantapää-Dahlqvist S, Berglin E, Stolt P and Padyukov L., 2004 - What precedes development of rheumatoid arthritis?.

Ann Rheum Dis, 63:28-31.

76

[46] Mattey D, Hutchinson D, Dawes P, et al., 2002 - Smoking and disease

severity in rheumatoid arthritis: association with polymorphism at the glutathione

S-transferase M1 locus. Arthritis Rheum, 46(3):640-6. [47] Heliövaara M, Aho K, Knekt P, Impivaara O, Reunanen A and Aromaa A., 2000 - Coffee consumption, rheumatoid factor, and the risk of rheumatoid

arthritis. Ann Rheum Dis, 59(8):631-5. [48] Gorman J., 2006 - Smoking and rheumatoid arthritis: another reason to just

say no. Arthritis Rheum, 54(1):10-3. [49] Silman A and Pearson J., 2002 - Epidemiology and genetics of

rheumatoid arthritis. Arthritis Res, 4 (suppl 3):S265-S272.

[50] Klareskog L, Padyukov L, Lorentzen J and Alfredsson L., 2006 -

Mechanisms of disease: Genetic susceptibility and environmental triggers in the

development of rheumatoid arthritis. Nat Clin Pract Rheumatol, 2(8):425-33.

[51] Olsson A, Skogh T, Axelson O and Wingren G., 2004 - Occupations and

exposures in the work environment as determinants for rheumatoid arthritis.

Occup Environ Med, 61(3):233-8.

[52] Skogh T., 2005 - Does a positive anti-CCP test identify a distinct arthritis

entity?. Arthritis Res Ther, 7(6):230-232.

[53] Klareskog L, Padyukov L, Rönnelid J and Alfredsson L., 2006 - Genes,

environment and immunity in the development of rheumatoid arthritis. Curr Opin

Immunol, 18(6):650-5.

77

[54] Hayer S, Tohidast-Akrad M, Haralambous S, et al., 2005 - Aberrant

expression of the autoantigen heterogeneous nuclear ribonucleoprotein-A2

(RA33) and spontaneous formation of rheumatoid arthritis-associated anti-RA33

autoantibodies in TNF-alpha transgenic mice. J Immunol, 175(12):8327-36.

[55] van der Helm-van Mil A, Verpoort K, Breedveld F, Huizinga T, Toes R, and de Vries R., 2006 - The HLA–DRB1 Shared Epitope Alleles Are Primarily a

Risk Factor for Anti–Cyclic Citrullinated Peptide Antibodies and Are Not an

Independent Risk Factor for Development of Rheumatoid Arthritis. Arthritis

Rheum, 54(4):1117-1121.

[56] Bläss S, Engel J and Burmester G., 1999 - The immunologic homunculus

in rheumatoid arthritis. Arthritis Rheum, 42(12):2499-506.

[57] Sandhu V and Isenberg D., 2006 - The Practical Application of Biological

therapies in rheumatoid arthritis. (http://www.touchbriefings.com/pdf/1692/Isenberg). Last accessed in

25/10/2007

[58] Anderson D., 2004 - TNF inhibitors: a new age in rheumatoid arthritis

treatment. Am J Nurs, 104(2):60-8.

[59] Choy E and Panayi G., 2001 – Cytokine pathways and joint inflammation

in rheumatoid arthritis. N Engl J Med, 344(12):907-916.

[60] Harris E., 2003 - The changing dimensions of rheumatoid arthritis and its

treatment. Bull World Health Organ, 81(9):631.

[61] Ritva Peltomaa., 2002 – Outcome of early rheumatoid arthritis with special

reference to early institution of drug treatment. (Dissertion). Helsinki University

Central Hospital, Finland.

78

[62] Hanjani A., 2000 - Genetic factors associated with disease severity in

Rheumatoid Arthritis. (Thesis). The university of British Columbia, Canada.

[63] http://patients.uptodate.com (last accessed in 25/10/2007)

[64] Maksimowicz-McKinnon K and Wilke W, 2004 – Rheumatoid Arthritis. In:

The Disease Management Project. Florida

http://www.clevelandclinicmeded.com/medicalpubs/.../rheumarth/rheumarth. (Last accessed in 25/10/2007). [65] Khurana R and Berney S., 2005 - Clinical aspects of rheumatoid arthritis.

Pathophysiology, 12:153–165.

[66] Brooks P., 2006 - Rheumatoid arthritis: aetiology and clinical

features. Medicine, 34: 379-382. [67] Feinstein D and Brent L., 2006 - The complexity of the differential

diagnosis for the inflammatory arthritides.

(www.postgradmed.com (last accessed in 25/10/2007).

[68] Meier E., 2006 - The Clinical Picture of Rheumatoid Arthritis. ELiA journal,

(www.phadia.com/upload/Autoimmunity/EliA%20Journal/Files/EJ_E_106. (last

accessed in 25/10/2007).

[69] Bjornadal L, Baecklund E, Yin L, Granath F, Klareskog L and Ekbom A., 2002 - Decreasing Mortality in Patients with Rheumatoid Arthritis: Results

from a Large Population Based Cohort in Sweden, 1964-95. J Rheumatol,

29:906-12.

[70] Liang K, Liang K, Matteson E, McClelland R, Christianson T and Turesson C., 2006 - Incidence of Noncardiac Vascular Disease in Rheumatoid

79

Arthritis and Relationship to Extraarticular Disease Manifestations. Arthritis

Rheum, 54(2):642-8. [71] Doran M, Pond G, Crowson C, O’Fallon W and Gabriel S., 2002 -

Trends in Incidence and Mortality in Rheumatoid Arthritis in Rochester,

Minnesota, Over a Forty-Year Period. Arthritis Rheum, 46(3):625-631.

[72] Ekström K, Hjalgrim H, Brandt L, Baecklund E, Klareskog L, Ekbom A

and Askling J. 2003 - Risk of malignant lymphomas in patients with

rheumatoid arthritis and in their first-degree relatives. Arthritis Rheum,

48(4):963-70.

[73] Symmons D, Mathers C and Pfleger B., 2000 - The global burden of

rheumatoid arthritis in the year 2000.

(www.who.int/entity/healthinfo/statistics/bod_rheumatoidarthritis) last accessed

in 25/10/2007).

[74] Symmons D., 2002 - Epidemiology of rheumatoid arthritis: determinants of

onset, persistence and outcome. Best Pract Res Clin Rheumatol, 16(5):707-22. [75] Solanki K, Spellerberg M, Chapman P, Moller P and O'Donnell J., 2004

- Anti-cyclic citrullinated antibodies: complementary to IgM rheumatoid factor in

the early diagnosis of rheumatoid arthritis. N Z Med J, 117(1203):1-5. [76] O'Dell J., 2004 - Therapeutic Strategies for Rheumatoid Arthritis. N Z Med

J, 350(25):2591-2602. [77] Harrison B, Silman A, Symmons D, Hulsemann J and Zeidler H., 2000 -

Diagnostic evaluation of classification criteria for RA and reactive arthritis. Ann

Rheum Dis, 59:397-398.

80

[78] Kary S, Fritz J, Scherer H and Burmester G., 2004 - Do we still miss the

chance of effectively treating early rheumatoid arthritis? New answers from a

new study. Rheumatology, 43:819–820.

[79] Saraux A, Berthelot J, Chalès G, et al., 2001 - Ability of the American

College of Rheumatology 1987 criteria to predict rheumatoid arthritis in patients

with early arthritis and classification of these patients two years later. Arthritis

Rheum, 44(11):2485-91.

[80] Vasishta A., 2002 - Diagnosing early-onset rheumatoid arthritis : The role

of anti-CCP antibodies. Am Clin Lab, 21(7):34-6.

[81] Tsavaris N, Mavragani C and Pikazis D., 2007 - Rheumatoid arthritis:

correlation between rheumatoid factor levels and CA-125 tumour marker

elevation. Ann Rheum Dis, 66(7):980.

[82] Elaine Moore., 2006 - Rheumatoid Factor: Diagnosing Rheumatoid Arthritis

and Related Disorders.

http:// autoimmunedisease.suite101.com/article.cfm/rheumatoidfactortests (last

accessed in 28/10/2007).

[83] Halldórsdóttir H, Jónsson T, Thorsteinsson J and Valdimarsson H., 2000 - A prospective study on the incidence of rheumatoid arthritis among

people with persistent increase of rheumatoid factor. Ann Rheum Dis, 59(2):149-

51.

[84] Tešija – Kuna A., 2006 - Laboratory standards in diagnosis and therapy

monitoring of rheumatoid arthritis. eJIFCC,17(3) Paper 8.

[85] Palosuo T, Tilvis R, Strandberg T and Aho K., 2003 - Filaggrin related

antibodies among the aged. Ann Rheum Dis, 62(3):261-3.

81

[86] Sharif S, Eghbal S, Gharibdoost F, et al., 2007 - Comparative study of

anti-CCP and RF for the diagnosis of rheumatoid arthritis. APLAR J Rheumatol, 10 (2): 121-124. [87] Suzuki K, Sawada T, Murakami A, et al., 2003 - High diagnostic

performance of ELISA detection of antibodies to citrullinated antigens in

rheumatoid arthritis. Scand J Rheumatol, 32(4):197-204. [88] http://www.rheumatology.org/publications/hotline/1003anticcp.asp (last accessed in 28/10/2007) [89] http://snp.com.au/publications/pdf/doct/info/Connective%20Tissue.pdf (last accessed in 28/10/2007) [90] Steiner G and Smolen J., 2002 - Autoantibodies in rheumatoid arthritis

and their clinical significance. Arthritis Res, 4:S1-S5. [91] Koivula M, Aman S, Karjalainen A, Hakala M and Risteli J., 2005 - Are

there autoantibodies reacting against citrullinated peptides derived from type I

and type II collagens in patients with rheumatoid arthritis?. Ann Rheum Dis,

64(10):1443-50. [92] Riedemann J, Muñoz S and Kavanaugh A., 2005 - The use of second

generation anti-CCP antibody (anti-CCP2) testing in rheumatoid arthritis--a

systematic review. Clin Exp Rheumatol, 23(5 Suppl 39):S69-76. [93] Nogueira L, Sebbag M, Vincent C, et al., 2001 - Performance of two

ELISAs for antifilaggrin autoantibodies, using either affinity purified or

deiminated recombinant human filaggrin, in the diagnosis of rheumatoid arthritis. Ann Rheum Dis, 60(9):882-7.

82

[94] Chapuy-Regaud S, Nogueira L, Clavel C, Sebbag M, Vincent C and Serre G., 2005 - IgG subclass distribution of the rheumatoid arthritis-specific

autoantibodies to citrullinated fibrin. Clin Exp Immunol,139(3):542-50. [95] Vossenaar E, Radstake T, van der Heijden A, et al., 2004 - Expression

and activity of citrullinating peptidylarginine deiminase enzymes in monocytes

and macrophages. Ann Rheum Dis, 63(4):373-81. [96] Masson-Bessière C, Sebbag M, Girbal-Neuhauser E, Nogueira L, Vincent C, Senshu T and Serre G., 2001 - The major synovial targets of the

rheumatoid arthritis-specific antifilaggrin autoantibodies are deiminated forms of

the alpha- and beta-chains of fibrin. J Immunol, 166(6):4177-84. [97] Vossenaar E, Després N, Lapointe E, et al ., 2004 - Rheumatoid arthritis

specific anti-Sa antibodies target citrullinated vimentin. Arthritis Res Ther,

6(2):R142-50. [98] Vossenaar E and Robinson W., 2005 - Citrullination and autoimmune

disease: 8th Bertine Koperberg meeting. Ann Rheum Dis, 64:1513–1515.

[99] van Boekel M, Vossenaar E, van den Hoogen F and van Venrooij W., 2002 - Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and

diagnostic value. Arthritis Res, 4(2):87-93.

[100] Vossenaar E and van Venrooij W., 2004 - Citrullinated proteins: sparks

that may ignite the fire in rheumatoid arthritis. Arthritis Res Ther, 6(3):107-11.

[101] Gregersen P., 1999 - Genetics of rheumatoid arthritis: confronting

complexity. Arthritis Res, 1(1):37-44.

83

[102] Hill J, Southwood S, Sette A, Jevnikar A, Bell D and Cairns E., 2003 - Cutting edge: the conversion of arginine to citrulline allows for a high-affinity

peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401

MHC class II molecule. J Immunol, 171(2):538-41. [103] Goldbach-Mansky R, Lee J, McCoy A, et al., 2000 - Rheumatoid arthritis

associated autoantibodies in patients with synovitis of recent onset. Arthritis

Res, 2(3):236-43.

[104] Vossenaar E, Zendman A, Van Venrooij W., 2004 - Citrullination, a

possible functional link between susceptibility genes and rheumatoid arthritis. Arthritis Res Ther, 6(1):1-5. [105] Suzuki A, Yamada R, Chang X, et al., 2003 - Functional haplotypes of

PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are

associated with rheumatoid arthritis. Nat Genet, 34(4):395-402.

[106] van Venrooij W, Zendman A, Nijenhuis S, Vossenaar E, Wiik A, and Pruijn G., 2004- Antibodies to cyclic citrullinated peptides (anti-CCP) are

specific for the diagnosis and prognosis of (early) rheumatoid arthritis. www.phadia.com/upload/Autoimmunity/EliA%20Journal/Files/Web_EJE_2_04.pdf (Last accessed in 28/10/2007). [107] Hoffman I, Peene I, Pottel H, et al., 2005 - Diagnostic performance and

predictive value of rheumatoid factor, anti-citrullinated peptide antibodies, and

the HLA shared epitope for diagnosis of rheumatoid arthritis. Clin Chem,

51(1):261-3. [108] Lam G., 2006 - Rounds 4: Citrullinated Proteins, Peptidylarginine

Deiminase (PAD), and Rheumatoid Arthritis.

84

http://www.hopkinsarthritis.org/rheumrounds/citrullinated_proteins_rheumrounds (last accessed in 28/10/2007). [109] Khosla P, Shankar S and Duggal L., 2004 – Anti CCP antibodies in

rheumatoid arthritis. J Indian Rheumatol Assoc, 12: 143 -46.

[110] Kroot E, de Jong B, van Leeuwen M, et al., 2000 - The prognostic value

of anti-cyclic citrullinated peptide antibody in patients with recent-onset

rheumatoid arthritis. Arthritis Rheum, 43(8):1831-5.

[111] Vencovský J, Machácek S, Sedová L, Kafková J, Gatterová J, Pesáková V and Růzicková S., 2003 - Auto antibodies can be prognostic

markers of an erosive disease in early rheumatoid arthritis. Ann Rheum Dis,

62(5):427-30.

[112] Visser H, le Cessie S, Vos K, Breedveld F and Hazes J., 2002 - How to

diagnose rheumatoid arthritis early: a prediction model for persistent (erosive)

arthritis. Arthritis Rheum, 46(2):357-65.

[113] Bombardieri M, Alessandri C, Labbadia G, et al., 2004 - Role of anti-

cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid

arthritis from patients with chronic hepatitis C infection-associated polyarticular

involvement. Arthritis Res Ther, 6(2):R137-41. [114] Sène D, Ghillani-Dalbin P, Limal N, Thibault V, van Boekel T, Piette J and Cacoub P., 2006 - Anti-cyclic citrullinated peptide antibodies in hepatitis C

virus associated rheumatological manifestations and Sjogren's syndrome. Ann

Rheum Dis, 65(3):394-7. [115] Mediwake R, Isenberg D, Schellekens G and van Venrooij W., 2001 - Use of anti-citrullinated peptide and anti-RA33 antibodies in distinguishing

85

erosive arthritis in patients with systemic lupus erythematosus and rheumatoid

arthritis. Ann Rheum Dis, 60(1):67-8. [116] Alessandri C, Bombardieri M, Papa N, Cinquini M, Magrini L, Tincani A and Valesini G., 2004 - Decrease of anti-cyclic citrullinated peptide antibodies

and rheumatoid factor following anti-TNF therapy (infliximab) in rheumatoid

arthritis is associated with clinical improvement. Ann Rheum Dis, 63(10): 1218–

1221.

[117] Langguth D and Wong R., 2006 - Antibodies to cyclic citrullinated

peptides: how they assist in the diagnosis of rheumatoid arthritis. Aust Prescr,

29:162–4.

[118] http:// autoimmunedisease.suite101.com/blog.cfm/1549 - 29k (last

accessed in 28/10/2007)

[119] Hueber W, Kidd B, Tomooka B, et al., 2005 - Antigen microarray

profiling of autoantibodies in rheumatoid arthritis. Arthritis Rheum, 52(9):2645-

55. [120] Hueber W, Utz P and Robinson W., 2003 - Autoantibodies in early

arthritis: advances in diagnosis and prognostication. Clin Exp Rheumatol, 21(5

Suppl 31):S59-64. [121] Morris R., New, Highly Specific Autoantibody Markers in RA Present

Status and Future Prospects www.rdlinc.com/pdf/articles/AutoantibodiesRA-DrMorris-10-5-06_4.pdf (last

accessed in 28/10/2007) [122] Jonas B and Deane K., 2006 - The role of diagnostic studies in the

evalution and treatment of early rheumatoid arthritis: focus on autoantibodies,

inflammatory markers, and genetic testing.

86

www.med.unc.edu/cme/documents/2800 monograph_Dean [for web]. (last

accessed in 28/10/2006). [123] Lindqvist E, Eberhardt K, Bendtzen K, Heinegård D and Saxne T., 2005 - Prognostic laboratory markers of joint damage in rheumatoid arthritis.

Ann Rheum Dis, 64(2):196-201 [124] James D, Young A, Kulinskaya E, Knight E, Thompson W, Ollier W and Dixey J., 2004 - Orthopaedic intervention in early rheumatoid arthritis.

Occurrence and predictive factors in an inception cohort of 1064 patients

followed for 5 years. Rheumatology (Oxford), 43(3):369-76.

[125] St Clair E, van der Heijde D, Smolen J, et al., 2004 - Combination of

infliximab and methotrexate therapy for early rheumatoid arthritis: a randomized,

controlled trial. Arthritis Rheum, 50(11):3432-43.

[126] Chen H, Lin K, Chen C, et al., 2006 - The effect of etanercept on anti-

cyclic citrullinated peptide antibodies and rheumatoid factor in patients with

rheumatoid arthritis. Ann Rheum Dis, 65(1):35-9. [127] Buch M, Seto Y, Bingham S, Bejarano V, Bryer D, White J, and Emery P., 2005 - C-Reactive Protein as a Predictor of Infliximab Treatment Outcome in

Patients With Rheumatoid Arthritis. Arthritis Rheum, 52(1):42-48. [128] Antti Kuuliala., 2006 – Studies on immune activation in rheumatoid

arthritis and reactive arthritis. (dissertion), university of Helsinki, Finland.

[129] O’Dell J., 2002 - Treating Rheumatoid Arthritis Early: A Window of

Opportunity?. Arthritis Rheum, 46(2): 283-285. [130] Suresh E and Lambert C., 2005 - Combination treatment strategies in

early rheumatoid arthritis. Ann Rheum Dis, 64:1252-1256.

87

[131] Koivula M., 2006 – Autoantibodies binding citrullinated type I and II

collagens in rheumatoid arthritis. (Dissertion). University of Oulu, Finland.

[132] Kumar A and Marwaha V ., 2003 - New Therapies for Rheumatoid

Arthritis. MJAFI, 59: 90-92.

[133] Pincus T., 2005 - A 3-page standard protocol to evaluate rheumatoid

arthritis (SPERA): Efficient capture of essential data for clinical trials and

observational studies. Clin Exp Rheumatol, 23, (Suppl. 39):114-119.

[134] Dhillon V and Gray M., 2006 - Anti-CCP2 may replace rheumatoid factor

test in rheumatoid arthritis. J R Coll Physicians Edinb, 36:220–221.

[135] Choi S, Lim M, Shin D, Park J and Shim S., 2005 - Diagnostic

performances of anti-cyclic citrullinated peptides antibody and antifilaggrin

antibody in Korean patients with rheumatoid arthritis. J Korean Med Sci, 20:

473-8.

[136] Kamoun M., 2005 - Diagnostic performance and predictive value of anti-

citrullinated peptide antibodies for diagnosis of rheumatoid arthritis: toward more

accurate detection?. Clin Chem, 51(1):12-3.

[137] Vasiliauskiene L, Wiik A and Høier-Madsen M., 2001 - Prevalence and

clinical significance of antikeratin antibodies and other serological markers in

Lithuanian patients with rheumatoid arthritis. Ann Rheum Dis, 60(5):459-66. [138] Hill J, Al-Bishri J, Gladman D, Cairns E and Bell D., 2006 - Serum

autoantibodies that bind citrullinated fibrinogen are frequently found in patients

with rheumatoid arthritis. J Rheumatol, 33(11):2115-9.

88

[139] Forslind K, Ahlmén M, Eberhardt K, Hafström I and Svensson B., 2004 - Prediction of radiological outcome in early rheumatoid arthritis in clinical

practice: role of antibodies to citrullinated peptides (anti-CCP). Ann Rheum Dis,

63:1090-1095. [140] Nielen M, van der Horst A, van Schaardenburg D, et al., 2005 - Antibodies to citrullinated human fibrinogen (ACF) have diagnostic and

prognostic value in early arthritis. Ann Rheum Dis, 64(8):1199-204. [141] Lee D and Schur P., 2003 - Clinical utility of the anti-CCP assay in

patients with rheumatic diseases. Ann Rheum Dis, 62:870-874.

[142] Shankar S, Grover R and Handa R., 2006 - Role of anti cyclic

citrullinated peptide antibodies in erosive disease in patients with rheumatoid

arthritis. Indian J Med Res, 124(6):689-96.

[143] Avouac J, Gossec L and Dougados M., 2006 - Diagnostic and predictive

value of anti-cyclic citrullinated protein antibodies in rheumatoid arthritis: a

systematic literature review. Ann Rheum Dis, 65(7):845-51.

[144] Bas S, Perneger T, Seitz M, Tiercy J, Roux-Lombard P and Guerne P., 2002 - Diagnostic tests for rheumatoid arthritis: comparison of anti-cyclic

citrullinated peptide antibodies, anti-keratin antibodies and IgM rheumatoid

factors. Rheumatolog, 41: 809-814. [145] Schellekens G, Visser H, de Jong B, van den Hoogen F, Hazes J, Breedveld F and van Venrooij W., 2000 - The diagnostic properties of

rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis

Rheum, 43(1):155-63.

89

[146] Reckner Olsson A, Skogh T and Wingren G., 2001 - Comorbidity and

lifestyle, reproductive factors, and environmental exposures associated with

rheumatoid arthritis. Ann Rheum Dis, 60(10):934-9. [147] Vannini A, Cheung K, Fusconi M, et al., 2007 - Anti-cyclic citrullinated

peptide positivity in non-rheumatoid arthritis disease samples: citrulline-

dependent or not?. Ann Rheum Dis, 66:511-516.

[148] Wu R, Shovman O, Zhang Y, Gilburd B, Zandman-Goddard G and Shoenfeld Y., 2007 - Increased prevalence of anti-third generation cyclic

citrullinated peptide antibodies in patients with rheumatoid arthritis and CREST

syndrome. Clin Rev Allergy Immunol, 32(1):47-56.

[149] Hueber W, Tomooka B, Zhao X, et al., 2007 - Proteomic analysis of

secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is

associated with up regulation of proinflammatory cytokines. Ann Rheum Dis,

66(6):712-9. [150] Dubucquoi S, Solau-Gervais E, Lefranc D, et al., 2004 - Evaluation of

anti-citrullinated filaggrin antibodies as hallmarks for the diagnosis of rheumatic

diseases. Ann Rheum Dis, 63(4):415-9. [151] Bas S, Perneger T, Kunzle E and Vischer T., 2002 - Comparative study

of different enzyme immunoassays for measurement of IgM and IgA rheumatoid

factors. Ann Rheum Dis, 61(6):505-10.

[152] Tampoia M, Brescia V, Fontana A, Maggiolini P, Lapadula G and Pansini N., 2005 - Anti-cyclic citrullinated peptide autoantibodies measured by

an automated enzyme immunoassay: analytical performance and clinical

correlations. Clin Chim Acta, 355(1-2):137-44.

90

[153] Herold M, Boeser V, Russe E and Klotz W., 2005 - Anti-CCP: history

and its usefulness. Clin Dev Immunol, 12(2):131-5. [154] De Rycke L, Peene I, Hoffman I, et al., 2004 - Rheumatoid factor and

anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value,

associations with radiological progression rate, and extra-articular

manifestations. Ann Rheum Dis, 63(12):1587-93.

[155] Silveira I and Burlingame R, von Mühlen C, Bender A and Staub H., 2007 - Anti-CCP antibodies have more diagnostic impact than rheumatoid

factor (RF) in a population tested for RF. Clin Rheumatol, 26(11):1883-9.

[156] Nishimura K, Sugiyama D, Kogata Y, et al., 2007 - Meta-analysis:

diagnostic accuracy of anti-cyclic citrullinated peptide antibody and rheumatoid

factor for rheumatoid arthritis. Ann Intern Med, 146(11):797-808. [157] Alexiou I, Germenis A, Ziogas A, Theodoridou K and Sakkas L., 2007

- Diagnostic value of anti-cyclic citrullinated peptide antibodies in Greek patients

with rheumatoid arthritis. BMC Musculoskelet Disord, 20:8:37. [158] García-Berrocal B, González C, Pérez M, Navajo J, Moreta I, Dávila C and González-Buitrago J., 2005 - Anti-cyclic citrullinated peptide

autoantibodies in IgM rheumatoid factor-positive patients. Clin Chim Acta,

354(1-2):123-30.

[159] Grootenboer-Mignot S, Nicaise-Roland P, Delaunay C, Meyer O, Chollet-Martin S and Labarre C., 2004 - Second generation anti-cyclic

citrullinated peptide (anti-CCP2) antibodies can replace other anti-filaggrin

antibodies and improve rheumatoid arthritis diagnosis. Scand J Rheumatol,

33(4):218-20.

91

[160] Costenbader K and Karlson E., 2006 - Cigarette smoking and

autoimmune disease: what can we learn from epidemiology?. Lupus, 15(11):

737–45. [161] Westwood O, Nelson P and Hay F., 2006 - Rheumatoid factors: what's

new?. Rheumatology (Oxford), 45(4):379-85.

[162] Vittecoq O, Incaurgarat B, Jouen-Beades F, et al., 2004 -

Autoantibodies recognizing citrullinated rat filaggrin in an ELISA using

citrullinated and non-citrullinated recombinant proteins as antigens are highly

diagnostic for rheumatoid arthritis. Clin Exp Immunol, 135(1):173-80.

[163] Tamai M, Kawakami A, Iwanaga N, et al., 2006 - Examination of IgM

rheumatoid factor (IgM-RF) and anti-cyclic citrullinated peptide antibody (anti-

CCP Ab) in Japanese patients with palindromic rheumatism. Intern Med,

45(12):795-7.

[164] Fernández-Suárez A, Reneses S, Wichmann I, Criado R and Núñez A., 2005 - Efficacy of three ELISA measurements of anti-cyclic citrullinated

peptide antibodies in the early diagnosis of rheumatoid arthritis. Clin Chem Lab

Med, 43(11):1234-9.

[165] Nielen M, van Schaardenburg D, Reesink H, et al., 2004 - Specific

autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial

measurements in blood donors. Arthritis Rheum, 50(2):380-6. [166] Pruijn G, Vossenaar E, Drijfhout J, Venrooij W and Zendman A., 2005

- Anti-CCP Antibody Detection Facilitates Early Diagnosis and Prognosis of

Rheumatoid Arthritis. CRR, 1:1-7.

92

[167] Chou C, Liao H, Chen C, Chen W, Wang H and Su K., 2007 - The

Clinical application of Anti-CCP in rheumatoid arthritis and other rheumatic

diseases. Biomark Insights, 2:165–171.

[168] Gottenberg J, Mignot S, Nicaise-Rolland P, et al., 2005 - Prevalence of

anti-cyclic citrullinated peptide and anti-keratin antibodies in patients with

primary Sjögren's syndrome. Ann Rheum Dis, 64(1):114-7. [169] Lienesch D, Morris R, Metzger A, Debuys P and Sherman K., 2005 - Absence of cyclic citrullinated peptide antibody in nonarthritic patients with

chronic hepatitis C infection. J Rheumatol, 32(3):489-93. [170] Takasaki Y, Yamanaka K, Takasaki C, et al., 2004 - Anticyclic

citrullinated peptide antibodies in patients with mixed connective tissue disease. Mod Rheumatol, 14(5):367-75. [171] Caspi D, Anouk M, Golan I, et al., 2006 - Synovial fluid levels of anti-

cyclic citrullinated peptide antibodies and IgA rheumatoid factor in rheumatoid

arthritis, psoriatic arthritis, and osteoarthritis. Arthritis Rheum, 55(1):53-6. [172] Böckelmann R, Gollnick H and Bonnekoh B., 2006 - Anti-cyclic

citrullinated peptide antibodies in psoriasis patients without arthritis. Arthritis

Rheum, 54(5):1701-2. [173] Meyer O, Labarre C, Dougados M, et al., 2003 - Anticitrullinated

protein/peptide antibody assays in early rheumatoid arthritis for predicting five

year radiographic damage. Ann Rheum Dis, 62(2):120-6.

[174] Meyer O, Nicaise-Roland P, Santos M, et al., 2006 - Serial

determination of cyclic citrullinated peptide autoantibodies predicted five-year

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radiological outcomes in a prospective cohort of patients with early rheumatoid

arthritis. Arthritis Res Ther, 8(2):R40. [175] Vallbracht I, Rieber J, Oppermann M, Förger F, Siebert U and Helmke K., 2004 - Diagnostic and clinical value of anti-cyclic citrullinated peptide

antibodies compared with rheumatoid factor isotypes in rheumatoid arthritis. Ann

Rheum Dis, 63(9):1079-84. [176] Vittecoq O, Pouplin S, Krzanowska K, et al., 2003 - Rheumatoid factor

is the strongest predictor of radiological progression of rheumatoid arthritis in a

three-year prospective study in community-recruited patients. Rheumatology

(Oxford), 42(8):939-46. [177] Turesson C, Jacobsson L, Sturfelt G, Matteson E, Mathsson L and Rönnelid J., 2007 - Rheumatoid factor and antibodies to cyclic citrullinated

peptides are associated with severe extra-articular manifestations in rheumatoid

arthritis. Ann Rheum Dis, 66(1):59-64. [178] Alexiou L, Germenis A, Ziogas A, Koutroumbas A, Theodoridou K, Kontogianni A and Sakkas L., 2007 - Diagnostic value of anticyclic citrullinated

peptide antibodies in Greek patients with rheumatoid arthritis: association with

extra-articular manifestations. Arthritis Research & Therapy, 9(Suppl 3):P30.

[179] Korkmaz C, Us T, Kaşifoğlu T and Akgün Y., 2006 - Anti-cyclic

citrullinated peptide (CCP) antibodies in patients with long-standing rheumatoid

arthritis and their relationship with extra-articular manifestations. Clin Biochem,

39(10):961-5.

[180] Greiner A, Plischke H, Kellner H and Gruber R., 2005 - Association of

anti-cyclic citrullinated peptide antibodies, anti-citrullin antibodies, and IgM and

IgA rheumatoid factors with serological parameters of disease activity in

rheumatoid arthritis. Ann N Y Acad Sci, 1050:295-303.

94

[181] Nielen M, van Schaardenburg D, Reesink H, et al., 2006 - Simultaneous

development of acute phase response and autoantibodies in preclinical

rheumatoid arthritis. Ann Rheum Dis, 65(4):535-7. [182] Aldifran Ferreira da S, MATOS, Napoleão A, Áurea Maria Santana L., 2006 - Association of anti-cyclic citrullinated peptide antibody and severe

rheumatoid arthritis. Rev. Bras. Reumatol, 46(3):165-173. [183] Nielen M, van Schaardenburg D, Reesink H, et al., 2004 - Increased

levels of C-reactive protein in serum from blood donors before the onset of

rheumatoid arthritis. Arthritis Rheum, 50(8):2423-7. [184] Berglin E, Johansson T, Sundin U, Jidell E, Wadell G, Hallmans G and Rantapää-Dahlqvist S., 2006 - Radiological outcome in rheumatoid

arthritis is predicted by presence of antibodies against cyclic citrullinated peptide

before and at disease onset, and by IgA-RF at disease onset. Ann Rheum Dis,

65(4):453-8. [185] Stropuvienë S, Lapinienë G, Redaitienë E, Kirdaitë G, Dadonienë J., 2005 - Rheumatoid arthritis markers: antibodies against citrullinated peptides. Acta Medica Lituanica , 12(30: 37-41. [186] Shovman O, Gilburd B, Zandman-Goddard G, Sherer Y, Orbach H, Gerli R and Shoenfeld Y., 2005 - The diagnostic utility of anti-cyclic citrullinated

peptide antibodies, matrix metalloproteinase-3, rheumatoid factor, erythrocyte

sedimentation rate, and C-reactive protein in patients with erosive and non-

erosive rheumatoid arthritis. Clin Dev Immunol, 12(3):197-202. [187] De Rycke L, Verhelst X, Kruithof E, Van den Bosch F, Hoffman I, Veys E and De Keyser F., 2005 - Rheumatoid factor, but not anti-cyclic citrullinated

95

peptide antibodies, is modulated by infliximab treatment in rheumatoid arthritis.

Ann Rheum Dis, 64; 299-302.

[188] Irigoyen P, Lee A, Wener M, et al., 2005 - Regulation of anti-cyclic

citrullinated peptide antibodies in rheumatoid arthritis: contrasting effects of

HLA-DR3 and the shared epitope alleles. Arthritis Rheum, 52(12):3813-8. [189] Berglin E, Padyukov L, Sundin U, et al., 2004 - A combination of

autoantibodies to cyclic citrullinated peptide (CCP) and HLA-DRB1 locus

antigens is strongly associated with future onset of rheumatoid arthritis. Arthritis

Res Ther, 6(4):R303-8. [190] van der Helm-van Mil A, Verpoort K, Breedveld F, Toes R and Huizinga T., 2005 - Antibodies to citrullinated proteins and differences in clinical

progression of rheumatoid arthritis. Arthritis Res Ther, 7(5):R949-58. [191] Kastbom A, Strandberg G, Lindroos A and Skogh T., 2004 - Anti-CCP

antibody test predicts the disease course during 3 years in early rheumatoid

arthritis (the Swedish TIRA project). Ann Rheum Dis, 63(9):1085-9. [192] Rönnelid J, Wick M, Lampa J, Lindblad S, Nordmark B, Klareskog L, van Vollenhoven R., 2005 - Longitudinal analysis of citrullinated protein/peptide

antibodies (anti-CP) during 5 year follow up in early rheumatoid arthritis: anti-CP

status predicts worse disease activity and greater radiological progression. Ann

Rheum Dis, 64(12):1744-9. [193] Bizzaro N, Mazzanti G, Tonutti E, Villalta D and Tozzoli R., 2001 - Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis. Clin Chem, 47(6):1089-93.

96

[194] Bongartz T, Cantaert T, Atkins S, et al., 2007 - Citrullination in extra-

articular manifestations of rheumatoid arthritis. Rheumatology (Oxford),

46(1):70-5. [195] Masi A, Aldag J and Chatterton R., 2006 - Sex Hormones and Risks of

Rheumatoid Arthritis and Developmental or Environmental Influences. Ann. N.Y.

Acad. Sci. 1069: 223–235. [196] Hutchinson D, Shepstone L, Moots R, Lear J and Lynch M., 2001 - Heavy cigarette smoking is strongly associated with rheumatoid arthritis (RA),

particularly in patients without a family history of RA. Ann Rheum Dis, 60(3):223-

7. [197] Nyhäll-Wåhlin B, Jacobsson L, Petersson I, Turesson C., 2006 - Smoking is a strong risk factor for rheumatoid nodules in early rheumatoid

arthritis. Ann Rheum Dis, 65(5):601-6.

[198] Uhlig T, Hagen KB, Kvien TK., 1999 - Current tobacco smoking, formal

education, and the risk of rheumatoid arthritis. J Rheumatol, 26(1):47-54.

[199] van Gaalen F, Linn-Rasker S, van Venrooij W, et al., 2004 - Autoantibodies to cyclic citrullinated peptides predict progression to rheumatoid

arthritis in patients with undifferentiated arthritis: a prospective cohort study. Arthritis Rheum, 50(3):709-15. [200] Svendsen A, Holm N, Kyvik K, Petersen P and Junker P., 2002 - Relative importance of genetic effects in rheumatoid arthritis: historical cohort

study of Danish nationwide twin population. BMJ, 324(7332):264-6 [201] www.sonoraquest.com/documents/rf_diag_panel_clinical_focus.pdf (last accessed in 29/11/2007).

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Annexes______________________________________________

Annex 1: RA patient's questionnaire.

ام . أنا الباحثة بشري سكيك يم فحص األجس تبيان من أجل عمل بحث حول تقي ذا اإلس ة ه أرجو المساعدة في تعبئ .المضادة ضد بروتين السيترولين في تشخيص التهاب المفاصل الروماتزمي في قطاع غزة

______ العمر________________________ اسم المريض

ٱ ال ٱ إذا آانت اإلجابة أنثي هل أنت حامل؟ نعم ٱ أنثي ٱذآر : الجنس

ٱ ال ٱهل الدورة الشهرية منتظمة؟ نعم

ٱ الجنوب ٱ الوسطي ٱ الشمال ٱغزة : مكان السكن محافظة

_________تاريخ سحب العينة___________ المهنة

هل ظهرت أعراض التهاب المفاصل الروماتزمي في أفراد عائلتك؟

ٱ ال ٱنعم

____ منذ حوالي� ال ٱنعم هل أنت مدخن؟

:تقييم التهاب المفاصل الروماتزمي في مراحله المبكرة . التي تظهر أو ظهرت عندكأمام األعراض األولية" " x ضع عالمة -1 اإلحساس باأللم في المفصلٱ تورم المفصلٱ تصلب المفصلٱ فقد وظيفة المفصلٱ احمرار وارتفاع درجة حرارة المفصلٱ ما هي أول المفاصل التي ظهرت فيها األعراض؟-2 المفاصل الصغيرة في اليدين أو القدمينٱ المفاصل الكبيرةٱ آالهماٱ ال أعلمٱ

_____________________ متي ظهرت هذه األعراض؟ _____________________ متي تم تشخيص المرض ؟

_____________________ متي بدأ معك الطبيب بالدواء المضاد لاللتهابات؟

:م1987تشخيص المرض حسب المعايير التي وضعتها الرابطة األمريكية للروماتزم عام دقيقة___ : ساعة ___ آم المدة؟ٱ ال ٱنعم اح تصلب المفاصل في الصب

___ آم عدد المفاصل؟ ٱ ال ٱنعم التهاب ثالثة مفاصل أو أآثر ٱ ال ٱنعم التهاب مفاصل اليدين و المعصمين

ٱ ال ٱنعم التهاب المفاصل يكون متماثل في الجسم ٱ ال ٱنعم وجود العقد الروماتزمية تحت الجلد

98

ٱ ال ٱنعم عامل الروماتزم موجب____ منذ متي بدأت؟ٱ ال ٱنعم التغيرات اإلشعاعية في اليدين و المعصمين

:قياس مدي شدة المرض : اصل أمراض خارج المف-1

ٱ ال ٱنعم أمراض أخرى خارج المفاصل؟ يهل ظهرت عندك أ : أمام المرض التي تعاني منه (x) آانت اإلجابة بنعم الرجاء وضع عالمة إذا

أمراض في الجهاز التنفسىٱ أمراض قلبيةٱوزن، " أعراض فيتلس ٱ دم البيضاء، انخفاض ال الحمي، انخفاض آرات ال " الطحال، تقرحات و صبغيات في الجلدلالدم، استئصا فقر ٱ تضخم الغدد الليمفاويةٱ خلل فى الكليةٱ التهاب األوعية الدمويةٱ أمراض عصبية و نفسيةٱ أمراض في الجهاز الهضميٱ جفاف الفمٱ جفاف العينٱ تشوهات في اليدينٱ ٱ ال ٱنعم هل قمت بأي عملية جراحية؟ -2

إذا آانت اإلجابة بنعم الرجاء تحديد نوع العملية؟_______________________________________________________________________

______________________________________________________________________________________________________________________________________________

______________________________________________________________________________________________________________________________________________

.ة اليومي وأعمالككات تأثير التهاب المفاصل علي نشاط-3

ال يؤثر ٱ تأثيره قليل ٱ تأثيره متوسط ٱ شديد يرهتأث ٱ

. ضع عالمة عمودية واحدة خالل الخط لتوضيح وضعك أو نشاطك الصحي الحالي-4

| | | متوسط أحسن حال أسوأ حال

100 50 % 0

:األشعة التشخيصية . أمام األشعة التي قمت بعملها" x" الرجاء وضع عالمة

أشعة اآس ٱ األشعة المقطعيةٱ األشعة الملونةٱ

99

األشعة التلفزيونية الفوق الصوتيةٱ األشعة المغناطيسيةٱ

:أمام التغيرات اإلشعاعية اآلتية " x" إذا قمت بعمل أي من هذه األشعة التشخيصية الرجاء وضع عالمة حدوث تآآل في العظام ٱ هشاشية العظامٱ تغيرات أخرىٱ

:الفحوصات المخبرية

: نتائج الفحوصات المخبرية

اسم الفحص النتيجة ESR CRP IgM RF ACCP2A

: التي تقوم بتناولهااألدوية بتناولها في الوقت الحالي؟أسماء األدوية التي تقوم أمام " x" ضع عالمة

)أآامول(مسكنات ٱ : مثل)NSAIDS(أدوية غير ستيرويدية مضاد لاللتهاب ٱ

ميثوترآساتٱ آلوروآوينٱ أفاراٱ

أدوية ستيرودية ٱ : أدوية أخري مثلٱ

أدوية ألمراض ضغط الدمٱ أدوية ألمراض الكلي ٱ أدوية ألمراض الصدريةٱ لقلب أدوية ألمراض اٱ

100

Annex 2: Approval to conduct the study from Helsinki committee in the Gaza Strip.

101

Annex 3: Official letters of request sent to hospitals general director of the Palestinian ministry of health to obtain approval to conduct the study in hospitals.

102