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TOM TAT LUAN AN english nghiên cứu nồng độ IL 6, IL 17 và TNF α huyết than

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INTRODUCTION
Rheumatoid arthritis (RA) is a systemic chronic inflammatory
disease characterized by joint swelling, joint tenderness, and
destruction of synovial joints, leading to severe disability and
premature mortality.
For a long time, T helper (Th) type 1 subsets have been known to
be an important in the pathogenesis of RA. The recent discovery of
another subset of Th cells, namely the Th17 lineage, is another
important Th cell lineage in the pathogenesis of RA.
Upon activation, Th17 cells secrete a variety of proinflammatory
cytokine, such as IL-17, IL-6 and TNF-α. IL-6, TNF-α drive a
chronic synovitis, a breakdown in articular cartilage and bone
erosions. Thus, levels of serum IL-6 maybe relate to the clinical and
sub-clinical characteristics of RA patients.
IL-17 cells activate macrophages, synovial fibroblasts, and
chondrocytes to produce cytokines, such as IL-6, TNF-α. IL-17
subsets appear to involve indirectly to the pathological patterns of
RA and thus levels of serum IL-17 maybe relate to the clinical and
sub-clinical characteristics of RA patients.

Therefore, the research “Evaluation of the serum levels of IL-6,
IL-17 and TNF-α in patients with RA’’ was carried out with two
objectives:
1. To study the circulating concentrations of IL-6, IL-17, TNF-α
and the clinical and sub-clinical characteristics of RA patients.


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2. To assess the relationships between the serum levels of IL-6,
IL-17, TNF-α and the clinical and sub-clinical characteristics of RA
patients.
*The scientific value
The curent study collaborate the roles of IL-6, TNF-α, especially
IL-17 in the pathogenesis of RA.
* The practical value
To determine the serum levels of IL-6, IL-17 and TNF-α of
Vietnamese RA patients, and the relations to clinical and sub-clinical
characteristics of patients with RA.
* New contributions of this doctoral thesis
- The mean and the median of serum IL-6 in RA patients were
19,06 ± 22,94 and 10,49 (pg/mL), respectively, those was significant
higher than that in healthy controls (p = 0,042, test Mann-Whitney).
- The mean and the median of serum TNF-α in RA patients were
2,37 ± 2,69 và 1,68 (pg/mL), respectively, those was significant
lower than that in healthy controls (p < 0,001, test Mann-Whitney).
- The mean and the median of serum IL-17 in RA patients were
0,59 ± 0,92 và 0,30 (pg/mL), respectively. There was no differences
of the median between RA patients and controls (p = 0,879, test
Mann-Whitney).
- The elevation of serum IL-17 tightly related to the changes of
serum IL-6 and TNF-α, with 36,60% of RA patients having the
elevated serum IL-17 level combining with the elevation of serum
IL-6 and/or TNF-α.


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The doctoral thesis arrangement: This current thesis includes 121
pages (without references and appendixes): Introduction: 02 pages,
Chapter 1 Overview: 29 pages, Chapter 2 Materials and methods: 21
pages, Chapter 3 Results: 30 pages, Chapter 4 Discussion: 36 pages,


Conclusion: 02 pages, Recommendations: 01 page. It also includes
30 tables, 14 graphs, 13 figures, and 150 references (14 Vietnamese
references and 136 English references)
CHAPTER1. OVERVIEW
1.1. Rheumatoid arthritis
1.1.1.

The concept of rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune chronic
inflammatory disease of unknown etiology marked by a symmetric,
peripheral polyarthritis.
1.1.2.

The clinical manifestations of rheumatoid arthritis

Articular features
The typical clinical manifestations of RA include morning
stiffness, peripheral polyarthritis of hands and feet, wrists, ankles,
knees, shoulders and hips. Patients may have chronic, irreversible
deformities of hands and feet in the established disease.
Extra-articular features
The most common extra-articular manifestations of RA include
mild fever, chronic anemia, and secondary Sjögren’s syndrome,
subcutaneous nodules which rarely occur.
Laboratory findings and joint imaging


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CRP blood level, erythrocyte sedimentation rate, RF, anti-CCP,
hand plain X-rays are performed for diagnosing of RA.
Musculoskeletal ultrasound joint and MRI are sometime used.
1.1.3.

Progressing in the diagnosis of rheumatoid arthritis

The diagnosing of RA is based on the 1987 ACR classfication
criteria. Recently, the 2010 ACR/EULAR classfication criteria for RA
is used to early diagnose of RA when patients are in initial disease.
1.1.4.

Treatment of rheumatoid arthritis

Medical treatment of RA includes nonpharmacologic therapy and
pharmacologic therapy. The pharmacologic therapy has had
prominent advances recently. Until now, there are two drug groups,
such as anti-inflammatory drugs, NSAIDs and glucocorticoids, and
DMARDs (Disease-Modifying antirheumatic Drugs).
DMARDs include nonbiologic DMARDs and biologic DMARDs.
MTX is the first choice of DMARDs and the cornerstone of RA
therapy.
1.1.5.

Assessment of rheumatoid arthritis activity

The assessment of disease activity has a very important role in
the prognosis of RA and is the key for choicing of DMARDs therapy.
This framework is performed frequently in the treatment of RA.
In addition to individual variables that include TJC28, SJC28,
morning stiffness, CRP, ESR; ACR and EULAR recommended using
composite indices for disease activity assessment include DAS,
DAS28, SDAI, CDAI.
1.2. Pathogenesis of rheumatoid arthritis


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RA is a chronic systemic autoimmune inflammatory caused by a
breakdown in immunologic self-tolerance that produces the
developing of autoantibodies that reponse to self-antigens.
In addition to the roles of antibodies, there is an evidence
supporting a role for self-reactive T cells that drive a synovial
chronic inflammation. T CD4+ cells include two subsets, Th1 and
Th17, both have a very important role in the RA’s pathogenesis.
Additionally, the deficiency of amount and functions of Tregs
induces to not

suppress self-reactive T cells reponsing to self-

antigens that also are mentioned in the pathogenesis of RA .
Th17 cells secret a variety of proinflammatory mediators, such as
IL-17, IL-21, IL-22, TNFα, IL-26, IL-6 and granulocytemacrophagecolony-stimulating factor (GM-CSF).
The proinflammatory cytokines include IL-1, IL-6, IL-17 and TNFα synergize in effects, and IL-17 appears a conductor that regulates the
connections between cytokines which combine with immune
complexes to drive a synovial chronic inflammatory response.
IL-1, IL-6, IL-17 and TNF-α also have an important role in
promoting

bone

resorption,

decreasing

bone

formation

via

RANK/RANKL/OPG system and stimulating a synovial pannus
developing.
1.3 The role of IL,-6, IL-17 and TNF-α in the pathogenesis of
rheumatoid
1.3.1. The role of IL-6, IL-17 and TNF-α in the synovial chronic
inflammation


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TNF-α, IL-1, IL-6, IL-17 are pivotal cytokines in the chronic
synovitis. They promote the influx of leukocytes into the synovial
microenvironment, neovascular developing and synovial fibroblast
proliferation.
1.3.2. The role of IL-6, IL-17 and TNF-α in the articular
breakdown
TNF-α, IL-1, IL-6, IL-17 enhance the expression of RANKL in
the microenvironment of joints which stimulates osteoclast
differentiation. TNF-α also suppresses the bone formation by
promoting of dickkopf-1 production.
TNF-α, IL-1, IL-6, and IL-17 stimulate the synovial fibroblasts to
secrete proteinases such as matrix metalloproteinases (MMPs) into
synovial fluid. In other words, IL-6 stimulates the production of
tissue inhibitor of MMP (TIMP).
1.4. Vietnames and international researches on IL-6, IL-17 and
TNF-α
1.4.1. International researches
1.4.1.1. The researchs on IL-6, IL-17 and TNF-α levels
The study results of Chung S.J. et al. (2011), do Prado A.D. et al.
(2016) show that serum IL-6 levels in RA patients were elevated than
that in healthy controls.
In concordance with these results, Metawi S.A. et al. (2011) and
Hanan M.A. et al. (2016) found that serum TNF-α was increased in
RA patients compared to healthy subjects.
The results of Hanan M.A. et al. (2016) and do Prado A. D. et al.
(2016) indicates that serum TNF-α in patients with RA was higher
than that in heathy controls.


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By constract, Tekeoglu I. et al. (2016) observed serum IL-6 in RA
being 14.32 ± 9.21 (pg/mL) and it had no differences compared to
healthy controls (13.45 ± 2.52 (pg/mL)).
Kokknen H. et al. (2010) found that the medium of serum IL-17
was 6.0 (pg/mL) which was significantly lower in RA patients
compared to healthy subjects (p = 6.1 x 10-5). Similarly, Selass O. et
al. (2015) found that serum TNF-α decreased in RA patients
compared to healthy controls.
1.4.1.2. The researchs on associations between IL-6, IL-17 and TNFα levels and the clinical and sub-clinical features of RA patients
Chung S.J. et al. (2011) observed that serum IL-6 had no
realationships with DAS28 ESR
Xia T. et al. (2015) found that serum IL-6 possitively correlated
with DAS28 ESR (r = 0,380; p < 0,05). do Prado A.D, et al (2016)
observed that serum TNF-α had no correlations with TJC28, SJC28,
serum CRP, ESR, DAS28 CRP and DAS28 ESR.
1.4.2. Vietnamese researches
Vietnam has had a lot of studies of RA so far, there have been,
however, a few researchs on the roles of serum IL-6, IL-17 and TNFα levels that were published.
Nguyen Ngoc Chau (2004) carried out a research on a few
immulogical factors of 32 RA patients. The results show that the
number of peripheral white blood cells and lymphocytes, serum
TNF-α and RF were significantly increased in RA patients compared
with controls (p < 0.001).
Recently, the study results of Vo Tam and Pham Thi Thu Tram
(2016) in 42 RA patients show that the median of serum IL-6 was


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36.4 (pg/mL) which was significantly higher than that in healthy
controls.
CHAPTER 2. MATERIALS AND METHODS
2.1. Materials
The study included 86 active RA patients and 30 healthy controls.
2.1.1. Selection criteria
* Patients:
+ Diagnosed with RA according to the 1987 ACR classification
criteria.
+ DAS28 CRP ≥ 2.6.
* Controls:
+ Healthy persons with matched-sex and the age from 35 to 50
years old.
2.1.2. Exclusion criteria
- Patients and controls do not agree to participate in the research.
- Patients have been treated by biologic DMARDs.
- Patients and controls with following diseases that affect to
serum cytokines: infectiuos diseases, malignant tumors, acute severe
internal diseases (acute myocardial infarction, acute stroke, acute
renal injury, congestive heart failure NYHA III, IV...)
2.1.3. Location and time of study
- Location: Department of Endocrinology and Rheumatology 103 Military Hospital.
- Time: from May 2012 to June 2016.
2.2. Methods
2.2.1. Study design
Analytical cross-sectional study with comparing before and after
treatment .


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2.2.2. Sampling methods: Convenience simple random sampling.
2.2.3. Research process
2.2.3.1. Before the standard treament
-

Patients

were

taken

history

and

physical

examinations to collect indicators according to a
medical record and determined serum IL-, IL-17 and
TNF-α levels.
-Treatment: patients were treated according to the
standard therapy that was recommended by ACR (the
American College of Rheumatoloy) and VRA (the
Vietnamese Rheumatology Association).
2.2.3.2.Fowlowing up and evalution after the standart
treatment
- Following up clinical indicators, routine laboratory,
serum IL-6, IL-17 and TNF-α after 03 months of the
standard therapy.
Controls:

assessed

indicators

according

to

a

medical record and determined serum IL-6, IL-17 and
TNF-α.
2.2.4. The methods of data collection
2.2.4.3. Concentration of IL-6, IL-17 and serum TNF-α
Serum IL-6, IL-17 and TNF-α levels were determined
by fluorescence covalent microbead immunosorbent assay - FCMIA,
accoding to the fluorescence immunoassay principle, using kit
Magnetic Luminex Sceening Assays, cataloge number: LXSAHM-3
(TNF-α, IL-6, IL-17A) that was manufactured and delivered by R&D
System (US), on Luminex 200 system that was manufactured and


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installed by Luminex Corporation (US). This testing procedure was
performed at the Department of Immunology - Vietnam Military Medical
University.
2.4. Cytokine test
2.4.1. Test materials
We collected 4 mL of venous blood of study subjects putting into the
pipe with no anticoagulant. The samples were brought to a laboratory
room in 30 minutes, warmed in the cabinets of 37 0C, centrifugal from
3500 to 5000 rounds per minute, separated the serum. The serum was
stored in the negative cabinets of -80 oC until determining serum
cytokines. To remove the broken red blood cell samples.
2.5. Criteria used in the study
2.5.1. The 1987 ACR classfication criteria
Rheumatoid Arthritis 1987 Revised Classification Criteria:
Morning stiffness (in/around joints, at least 1 hour before
maximal improvement)
Arthritis (swelling) of 3 or more joint areas
Symmetric arthritis (swelling, NOT bony overgrowth)
Arthritis of Hand joints (wrists, MCPs or PIPs)
Rheumatoid nodules
Rheumatoid factor (serum)
Radiographic changes (erosions and/or peri-articular osteopenia
in hand/wrist joints)
Requirements: ≥4 of the above 7 criteria. Criteria 1-4 must have
been present for at least 6 weeks
2.5.3. Criteria of RA patients’s disease activity: using DAS28 CRP


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2.5.4. Criteria of treatment’s targets
- Disease duration is less than 6 moths: remission
(DAS28 < 2,6)
- Disease duration is at leats 6 months: low disease
activity (DAS28 CRP < 3,2).
2.5.5.The EULAR criteria for treatment response of RA patients
2.5.6. Treatment regiment for RA patients: according to the
recommendations of ACR and VRA.
2.6. Data processing
All statistical analyses were performed using the statistical
package for social sciences (SPSS), version 18.0.
The cut-offs of serum cytokines were determined using ROC.
2.6. Diagrams of the study
86 RA patients

Post-treatment: 18 patients

30 healthy controls

Pre-treatment: 86 patients

- Assessing clinical and subclinical features
- Determining serum IL-6, IL-17, TNF-α levels

Determining serum IL-6,
IL-17, TNF-α levels

Statistical analysis
- Comparing serum IL-6, IL-17 and TNF-α between RA patients and controls.
- Clinical and subclinical features of RA patients

The first objective

The second objective
Conclusion

Diagram 2.6. Diagrams of the study
CHAPTER 3: RESULTS


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3.1. General characteristics of subjects
Table 3.1. Age, disease onset age, and disease duration
of rheumatoid arthritis patients
Parameters
Female (n = 75)
Male (n = 11)
Age
53.76 ± 7.09
51.27 ± 8.66
(year)
53.44 ± 7.30
Disease onset age
49.32 ± 9.38
48.33 ± 9.69
(year)
49.19 ± 9.37
Disease
duration
4.49 ± 5.58
2.94 ± 3.05
(year)
4.29 ± 5.34
Table 3.2 and 3.3. The most common age group and disease onset
age group were

50 - 59 with percentage of 47.6% and 38.4%,

respectively.
Graph 3.1 and 3.4. 20% of RA patients had a disease duration <6
months, and remainders were ≥6 months. The percentages of

RA

patients with pre-study treatment by glucocorticoid and DMARDs were
50.6% and 4.7%, respectively.
Graph 3.2 và 3.3. Comparing age, gender between rheumatoid
arthritis patients and controls
Patients
Controls

Characteristics
Age (year)
(Mean ± SD)
Sex
Male
(%)
Female

p

54.44 ± 7.30

41.6 ± 4.57

< 0.05*

12.79
87.21

13.33
86.67

> 0.05

*: Independent-Samples T Test
The mean of age in controls was lower than in patients (p < 0.05)
3.2 Clinical and subclinical charateristics and serum IL-6, IL-17
and TNF-α levels of rheumatoid arthritis patients


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3.2.1. Clinical and subclinical characteristics of rheumatoid
arthritis patients
Table 3.4 và 3.5. Clinical and subclinical characteristics
of rheumatoid arthritis patients
Parameters

Mean ± SD

Median

TJC28

10.52 ± 7.38

9.00

SJC28

14.13 ± 9.08

12.00

Morning stiffness (minutes)

37.25 ± 33.82

30.00

PtGA (cm)

7.16 ± 2.25

7.50

EGA (cm)

5.65 ± 1.92

6.00

The means of plasma CRP, the first-hour ESR, plasma anti-CCP,
red blood cell count, hemoglobin concentration, white blood cell
count were 68.20 ± 47.19 (mg/L), 81.10 ± 44.35 (mm/h), 109.02 ±
76.56 (IU/mL), 4.10 ± 0.52 (T/L), 113.03 ± 16.58 (g/L) and 9.18 ±
2.46 (G/L), respectively.
Table 3.6, appendix 4, appendix 5. The means of plasma CRP of
patients and controls were 68.20 ± 47.19 and 0.52 ± 0.36 (mg/L),
respectively. The means of the first-hour ESR of RA patients and
controls were 81.10 ± 44.35 and 7.76 ± 3.93 (mm/L), respectively.
The medians of plasma CRP and the first-hour ESR in RA patients
was higher than those in controls (p < 0.001). The means of
composite indices DAS28 CRP, DAS28 ESR, SDAI, CDAI were
6.19 ± 1.36; 6.66 ± 1.55; 44.18 ± 19.83; 37.27 ± 18.55, respectively.
Graph 3.5, 3.6, and appendix 6. The percentages of RA patients
with radiographic changes according Steinbroker criteria at stage 1, 2
and 3,4 were 62.20% and 30.80%, respectively; among them


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Steinbroker’s stage 2, 3, 4 were 55.10%. 79.50% of RA patients had
the DAS28 CRP > 5.1.
3.2.2. The features of serum IL-6, IL-17 and TNF-α levels of
rheumatoid arthritis patients
Table 3.8. Comparing serum IL-6, IL-17 and TNF-α levels between
Serum
cytokine
levels (pg/mL)
xx ± SD
IL-6
Median
xx
± SD
IL-17
Median
xx
± SD
TNF-α
Median

study subjects
Patients
Controls
n = 82
n = 30
19.06 ± 22.94
9.19 ± 8.43
10.49
7.18
0.59 ± 0.92
0.62 ± 0.94
0.30
0.27
2.37 ± 2.69
3.87 ± 2.11
1.68
3.69

p
0.042*
0.879
< 0.001*

*: Mann - Whitney Test

The median of serum IL-6 in RA patients was higher than that in
controls. By contrast, the median of serum TNF-α in RA patients was
lower than that in controls.
Table 3.9, graph 3.7, 3.8, 3.9. The characteristics of serum IL-6, IL17 and TNF of rheumatoid arthritis patients
Cytokine

Cut off value
(pg/mL)

Se

Sp

AUC

p

Elevated
(%)

IL-6

12.37

0.46

0.80

0.63

0.042

46.30

IL-17

0.21

0.67

0.44

0.51

0.88

56.10

TNF-α

2.87

0.70

0.79

0.74

< 0.001

20.70

Se: Sensitivity; Sp: Specificity; AUC: Area under the curve

The results of table 3.6 show that the percentages of RA patients
with the elevation of serum IL-6, IL-17 and TNF-α levels was 46.30;
56.01 và 20.70%, respectively.


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Graph 3.10, 3.11. Distribution of rheumatoid arthritis patients
according to serum cytokine levels
Normal
Increasing of the level of one
Distribution of
cytokine
rheumatoid arthritis
Increasing of the level of two
according to serum
cytokines
cytokine levels
Increasing of the level of three
cytokines
Normal
Increasing of the level of IL-17
Distribution of
rheumatoid arthritis Increasing of the level of IL-17
according to serum and IL-6 or/and TNF-α
IL-17 levels
Increasing of the level of IL-6
or TNF-α or IL-6 and TNF-α

26.80%
30.50%
35.40%
7.30%
26.80%
19.50
36.60%
17.10%

The percentage of RA patients with an elevated serum IL-17 was
56.10%, among them 36.60% of RA patients had an increased serum
IL-17 combining with the elevation of serum IL-6 or/and TNF-α.
Table 3.10. Comparing serum IL-6 và TNF-α levels according to the
Serum IL-17 levels
IL-6
(pg/mL)
TNF-α
(pg/mL)

xx ± SD
Median
xx ± SD
Median

serum IL-17 group
Normal
High
n = 36
n = 46
16.70 ± 23.19
20.68 ± 22.86
6.81
12.87
1.81 ± 2.53
2.81 ± 2.76
1.06
2.24

p
0.068*
< 0.05*

*: Mann-Whitney Test; IL-6: serum interleukin-6, TNF-α: Serum tumor
necrosis factor alpha

Graph 3.12, 3.13. The correlation between serum IL-6, IL-17 and
TNF-α levels
Cytokine
TNF-α

IL-6
r

IL-17
p

r

p


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0.233*

0.035

0.25*

0.023

*: Spearman's rank correlation coefficient

3.3. The relationships between serum IL-6, IL-17 and TNF-α
levels and clinical and subclinical characteristics of rheumatoid
arthritis patients
3.3.1. The relationships between serum IL-6, IL-17 and TNF-α
levels and clinical characteristics of rheumatoid arthritis patients
Table 3.11, 3.12, 3.13, 3.15, 3.16: There were no differences in
the medians of serum IL-6, IL-17 và TNF-α levels according to
TJC28, SJC28, DAS28 CRP, pre-study treatment by glucocorticoid
and disease duration.
Table 3.14 và graph 3.14: There were no differences in the
medians of TJC28, SJC28, PtGA, EGA, DAS28 according to serum
IL-6, IL-17 and TNF-α levels.
3.3.2. The relationships between serum IL-6, IL-17 and TNF-α
levels and subclinical features of rheumatoid arthritis
Table 3.17. The relationships between IL-6, IL-17 and TNF-α and
radiographic changes of RA patients with an elevated serum IL-17
Serum cytokine
Steinbroker
Steinbroker
p
(pg/mL)
stage 1, 2
stage 3, 4
IL-6
xx± SD 23.09 ± 18.97 14.53 ± 14.06 > 0.05*
Median
15.54
9.74
IL-17
xx± SD 0.89 ± 0.75
1.11 ± 1.34
> 0.05*
Median
0.60
0.70
Serum cytokine
Steinbroker
Steinbroker
p
(pg/mL)
stage 1, 2
stage 3, 4
TNF-α
xx± SD 3.34 ± 2.93
1.60 ± 1.48
0.05*
Median
2.67
1.06
IL −17
xx± SD
0.48 ± 0.99
0.75 ± 0.49
≤ 0.05*
TNF −α
Median
0.16
0.66


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*: Test Mann-Whitney

The results of table 3.17 show that the median of serum TNF-α in
RA patients with Steinbroker’s stage 1, 2 was higher than that in RA
patients with Steinbroker’s stage 3, 4 (p = 0.05).
Table 3.18. There were no differences in the medians of plasma
CRP, ESR after first hour, red blood cell count, hemoglobin
concentration, white blood cell count, plasma anti-CCP between two
RA patient groups with elevated or normal serum IL-6, IL-17 and
TNF-α.
Table 3.19. Serum IL-6, IL-17 and TNF-α were not correlated to
TJC28, SJC28, morning stiffness duration, PtGA, EGA, plasma CRP,
ESR after first hour, DAS28, SDAI, CDAI.
3.3.3. The relationships between serum IL-6, IL-17 and TNF-α
levels and the standard treatment of rheumatoid arthritis patients
Table

3.20.

The

means

of

age,

disease

duration,

methylprednisolone dosage were 54.28 ± 7.52 (year), 2.81 ± 3.41
(year), 11.33 ± 6.02 (mg/24h), respectively.
Table 3.21. The means of TJC28, SJC28, PtGA, EGA, plasma
CRP, DAS28 CRP were 14.11 ± 8.52; 11.61 ± 7.96; 6.94 ± 2.16; 5.79
± 1.63; 73.44 ± 56.28 (mg/L); 6.29 ± 1.26.
Table 3.22. The most DMARDs regiment was dualtherapy with
Methotrexate and Chloroquin (88.88%), in orther words, the
percentages of both Methotrexate monotherapy and tripletherapy
with Methotrexate, Sulfasalazine and Chloroquine were 5.56%.
Table 3.23, 3.24. The percentages of RA patients with good
response, moderate reponse and none were 12/16, 2/16 and 2/16


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patients, respectively. Only 3/17 of RA patients reached the goal
treatment after 03 months of the standard therapy.
Table 3.25. 3.26: There was significant decreases of TJC28,
SJC28, PtGA, EGA, plasma CRP, and composite indices such as
DAS28 CRP, SDAI, CDAI (p <0.01) after 03 months of the standard
therapy.
Table 3.27. The changes of serum cytokines of RA patients
after the standard therapy
Serum cytokine

T0

T3

p

IL-6

26.23 ± 31.25

10.94 ± 11.79

0.018*

IL-17

0.64 ± 1.04

0.79 ± 0.71

0.32*

TNF-α

2.97 ± 3.30

2.11 ± 2.18

0.163*

*: Test Wilcoxon, T0: the begining time of the standard therapy, T3: the
time after 03 months of the standard therapy

Table 3.28. The proportions of RA patients having decreases of
serum IL-6, IL-17 and TNF-α after the standard therapy were 13/18,
8/18, 13/18 patients, respectively.
Table 3.29. The mean of DAS28 CRP and the median of SDAI at
the T3 time point, of RA patients with serum IL-6 was less than 26.23
(pg/ml) at the T0 time point, were lower than those in RA patients with
serum IL-6 was more than or equal to 26.23 (pg/ml) at the T0 time
point (p < 0.05).
Table 3.23. The median of serum IL-6 at the T3 time point, of
RA patients with serum IL-6 was less than 26.23 (pg/ml) at the T0 time
point, was lower than that in RA patients with serum IL-6 was more
than or equal to 26.23 (pg/ml) at the T0 time point (p < 0.05).


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CHAPTER 4: DISCUSSION
4.1. General characteristics of investigated subjects
The percentages of femal and male of RA patients in this study
were 87.21% and 12.79%, respectively; the mean of age 53.44 ± 7.30
(year), and those were consistent with Hoang Trung Dung’s study
results (2011).
The mean of duration disease was 4.29 ± 5.34 (year), mostly from 6
months (80.0%), which was consistent with the results of Huu Thi
Chung (2008).
The percentages of patients using pre-study gluococorticoid and
DMARDs therapy were 4.70% and 50.6%, respectively.
The gender distribution of controls was consistent with RA patients;
the mean of controls’s age was 41.60 ± 4.57 (year). Although the mean
age was lower than that in patients (p < 0.05), however it appropriciated
for the onset disease age of RA which typically is at the moderate age,
and simultaneously decrease the patients with concomitent of
osteoarthritsis that maybe affects cytokine levels, such as IL-1β and
TNF-α.
4.2. Clinical and subclinical characteristics and serum IL-6, IL17 and TNF-α levels of rheumatoid arthritis patients
4.2.1. Clinical characteristics of rheumatoid arthritis patients
The means of TJC28 and SJC28 were 10.52 ± 7.38 and 14.13 ±
9.08, respectively; those were consistent with the results of Hoang
Trung Dung (2011). The means of PtGA, EGA were 7.16 ± 2.25 and
5.65 ± 1.92, respectively. PtGA was higher than EGA in this study
which was consistent with the ideas of Smolen S.J. et al. (2016).


20
The means of morning stiffness duration was 37.25 ± 33.82
(minute) was lower than that in the results of Hoang Trung Dung
(2011). Because the exact mechanisms of morning stiffness have not
understood, so it was not the same between other studies.
The mean of plasma CRP of RA patients was 68.20 ± 47.19
(mg/L), was higher than that in controls (p < 0.001). This result was
also higher than that in Hoang Trung Dung’s study (2011) due to the
levels of RA patient disease activity in this study were higher than
those of Hoang Trung Dung’s study (2011).
According to this study results, the mean of ESR after first hour
of RA patients was 81.10 ± 44.35 (mm/h), the median of it was
higher than that in controls (p < 0.001). This result was lower than
that in Hoang Trung Dung’s research (2011) but higher than that of
Huu Thi Chung (2009). Because study subjects were different
between other studies, so there were differences of age and gender
distribution.
The mean of DAS28 CRP was 6.19 ± 1.36; the percentage of RA
patients with DAS28 CRP > 5.1 was 79.5%.
Radiographic changes were at stages 1, 2 and stages 3, 4 (by
Steinbroker’s criteria) which were 69.20% and 30.80%, respectively,
among which 55.10% of RA patients were at stages 2, 3, 4 and only
44.90% of them were at stage 1.
4.2.2. Serum IL-6, IL-17 and TNF-α levels of rheumatoid arthritis
patients
Cut-off values of serum IL-6, IL-17 and TNF-α in RA patients
were 12.37, 0.21 and 2.87 (pg/mL). The percentages of RA patients


21
with high serum IL-6, IL-17 and TNF-α were 46.30, 56.01 and
20.70%, respectively.
7.30% of RA patients had a serum level elevation of all 3
cytokines (IL-6, IL-17 and TNF-α), 30.50% of RA patients had a
serum level elevation of 2 out of 3 cytokines, 30.50% of RA patients
had a serum level elevation of 1 out of 3 cytokines, and 26.80% of
RA patients had a normal serum levels of all 3 cytokines.
4.2.2.1. Characteristics of serum levels of IL-6
The mean and the median of serum IL-6 were 19.06 ± 22.94 and
10.49 (pg/mL). The median of serum IL-6 of RA patients was
statistically significant higher than that in controls (p = 0.042), in
which 46.30% of RA patients had a serum level elavation of IL-6.
This result was similar to that in the study of Vo Tam and Pham
Thi Thu Tram's study (2016) and do Prado A.D. et al. (2016).
However, the mechanism of a serum IL-6 elevation has not been
clear, thus according to the study of Tekeog˘lu I. et al. (2016), serum
IL-6 was not elevated compared to that in healthy controls.
Serum IL-6 was correlated to serum TNF-α (r = 0.233, p = 0.035).
This result was consistent with that in the study of Manicourt D.H. et
al. (1993). These results due to TNF-α could be a central role in RA
pathogenesis via stimulating of secreting cytokines, such as IL-6.
4.2.2.2. Characteristics of serum levels of TNF-α
The mean and the median of serum TNF were 2.37 ± 2.69 and
1.68 (pg/mL). The median of serum TNF-α in RA patients was
significantly lower than that in controls (p < 0.001), which was


22
similar to that in study of Selaas O. et al. (2015). The percentage of
RA patients who had a serum TNF-α elevation was 20.70%.
However, in the study of Kokkonen H. et al. (2010) with RA
patients’s mean disease duration was 7.7 ± 3.6 (month), the authors
found that serum TNF-α in 69 RA patients was not different from
that in sex-matched and age-matched controls (p = 0.223)
This study result also show that the median of serum TNF-α of
RA patients in the early period, with radiographic changes according
to Steinbroker’s criteria at stage 1, 2, which was higher than that in
the established period with Steibroker’stage at 3, 4 (p = 0.05).
Thus, the long-standing disease duration appears a factor to
decrease serum TNF-α in RA patients. Additionally, this study also
indicates that serum TNF-α was positively correlated to serum IL-6
và IL-17 levels.
4.2.2.3. Characteristics of serum levels of IL-17
The mean and the median of serum IL-17 in RA patients were
0.59 ± 0.92 and 0.30 (pg/mL), respectively. There were no
differences in the median of serum IL-17 between RA patients and
controls (p = 0.879), this result was consistent with the result of
Kokkonen H. et al. (2010). Additionally, the percentage of RA
patients with a serum IL-17 elevation was 56.10%.
In other words, serum IL-17 was positively correlated to serum
TNF-α (r = 0.25, p = 0.023) which was similar to that in the study of
Zhao P.W. et al. (2014).
Furthermore, the changes of serum IL-17 related to the changes of
serum IL-6 and TNF-α. In fact, Graph 3.11 show that the percentages


23
of RA patients had a serum IL-17 elevation combining with a serum
elevation of IL-6 and/or TNF-α which were 36.60%, compared to
17.10% of RA patients who had a serum elevation of IL-6, or TNF-α,
or IL-6 and TNF-α without the serum IL-17 elevation.
Simultaneously the results of table 3.10 show that the median of
serum TNF-α in RA patients with a serum IL-17 elevation was
significantly higher than that in patients with a normal serum IL-17
levels (p < 0.05).
Thus, this study shows that the serum IL-17 elevation tighly related to
the elevation of serum IL-6 and TNF-α levels. This results are consistent
with the perspective of Miossec P. (2007) on IL-17 that appears to be a
key role in the pathogenesis of RA.
4.3. The relationships between serum IL-6, IL-17, TNF-α levels
and clinical, subclinical characteristics of rheumatoid arthritis
patients
4.3.1. The relationships between serum IL-6 levels and clinical,
subclinical characteristics of rheumatoid arthritis patients
This study indicates that there were no relations between serum
IL-6 and clinical manefestations as well as subclinical features of RA
patients.
4.3.2. The relationships between serum IL-17 levels and clinical,
subclinical characteristics of rheumatoid arthritis patients
This study show that the median of serum TNF-α in RA patients
with radiogaphic changes according to Steinbroker’s criteria at stage
3, 4 was lower than that in RA patients with Steinbroker’s stage of 1,
2 (p = 0,05).


24
4.3.3 The relationships between serum TNF-α levels and clinical,
subclinical characteristics of rheumatoid arthritis
This study indicates that serum IL-17 was not related to clinical,
subclinical characteristics of RA patients. However, in the
established period of RA, the reduction of serum IL-17 tended to be
slower than that of TNF-α.
4.3.4. The relationships between serum IL-6, IL-17 and TNF-α
levels and the standard therapy of rheumatoid arthritis patients
4.3.4.2. The changes of serum IL-6, IL-17 and TNF-α levels after the
standard therapy in rheumatiod arthritis patients
After the 03-moth standard therapy, the median of serum IL-6
levels significantly reduced (p = 0.018), however, there were no
changes of the median of serum IL-17 and TNF-α levels. These
results were consistent with the study of Kremer J.M. et al. (2016),
which reveales a gradual reduction of serum IL-6 over the course of
the 36-month treatment.
CONCLUSIONS
Studying serum IL-6, IL-17 and TNF-α of 86 rheumatoid arthritis
patients and 30 healthy controls, the results show that:
1. Serum IL-6, IL-17, TNF-α levels and clinical, subclinical
characteristics of rheumatoid arthritis patients
1.1. Clinical and subclinical of rheumatoid arthritis patients
- The mean of disease duration were 4.29 ± 5.34 (year) with 80%
of RA patients were more than 6 months.


25
- The means of TJC28, SJC28, PtGA, EGA, plasma CRP, ESR
were 14.13 ± 9.08; 10.52 ± 7.38; 7.16 ± 2.25; 5.65 ± 1.92, 68.19 ±
47.19 (mg/L); 79.68 ± 44.37 (mm/h), respectively.
- The mean of DAS28 CRP was 6.19 ± 1.36 with 79.55% of RA
patients having DAS28 CRP was greater than 5.1.
- The percentage of RA patients with radiographic changes
according to Steinbroker criteria at the stage of 1, 2 and 3, 4 were
69.20 and 30.80%, respectively.
1.2. Serum IL-6, IL-17 and TNF-α levels of rheumatoid
arthritis patients
- The mean and the median of serum IL-6 in RA patients were
19.06 ± 22.94 and 10.49 (pg/mL), respectively. The median was
significantly higher than that in healthy controls (p = 0.042, MannWhitney test).
- The mean and the median of serum TNF-α in RA patients were
2.37 ± 2.69 and 1.68 (pg/mL), respectively. The median was
significantly lower than that in healthy controls (p < 0.001, MannWhitney test).
- The mean and the median of serum IL-17 in RA patients were
0.59 ± 0.92 and 0.30 (pg/mL), respectively. There was no differences
in the median between RA patients and controls (p = 0.879, MannWhitney test).
- Serum TNF-α was correlated to serum IL-6 and IL-17 with r, p
were 0.233; 0.035 and 0.25; 0.023, respectively, Spearman’s
correlation coefficient.


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