Association of sCD40 Level in Serum with Risk for Relapse in

Graves’ Disease

Trisia Lusiana Amir

, Dwi Anita Suryandari

, Fatimah Eliana

, Luluk Yunaini

and Dwi Yanti

Department of Physiotherapy, Faculty of Physiotherapy, Universitas Esa Unggul, Indonesia

Department of Medical Biology, Universitas Indonesia, Indonesia

Faculty of Medicine, University of YARSI, Indonesia

Biomedical Science, Faculty of Medicine, Universitas Indonesia, Indonesia

Keywords: sCD40, Graves’ Disease, Relapse, Remission, ELISA.

Abstract: CD40 is a transmembrane protein that influences the pathological of autoimmune disease, like Graves’

Disease (GD). Interaction with its ligand (CD40L) on the surface of CD4

T cells can enhance the humoral

immune response. Some studies indicated that GD patients have lower apoptosis in follicular cell. This

condition related to sCD40 level, which also can increase autoantibodies in GD patients. The aim of this

research was to find out the role of sCD40 as a marker on GD activity. This research was a case-control

study comparing 30 relapse patients and 30 non-relapse patients (remission) after treatment with an anti-

thyroid drug was terminated. The subject of this research was serum from both relapse and remission

patients, analyzed by ELISA method. Statistical analyzes was independent t-test, a two-tailed p-value less

than 0.05 was considered significant. The results, we found that sCD40 levels in serum (pg/ml) increased on

relapse patients (1.62± 0.17) compared to remission patients (1.46±0.14) and showed that sCD40 level in

serum was significantly different between relapse and remission (p<0.001). These results demonstrated that

elevated levels of sCD40 serum, associated with risk for relapse. So, sCD40 may be used as a marker of the

active stage of GD.

1 INTRODUCTION

Graves' disease (GD) is one of Autoimmune Thyroid

Disease (AITD), characterized by hyperthyroidism

and commonly found in women than men (Abbas,

2010 and Weetman, 2000). Based on Indonesia

Basic Health Survey (Riskesdas) 2013, the

prevalence of hyperthyroidism in Indonesia is 0.4%

with the highest in Jakarta as much as 0.7%

(Kemenkes, 2015). America is about 1% and

Caucasian population 0.5-2% (McLeod, 2012).

GD causes thyrotoxicosis, which increases the

thyroid hormone as the impact of the body's

mechanisms failure to self-tolerance, then

autoimmune reactions occur. Thyroid Stimulating

Hormone Receptor (TSHR) is an antigen that plays a

role in stimulating the formation of antibodies in the

body. These antibodies can bind and activate the

thyrotropin receptor and cause hypertrophy and

hyperplasia of the thyroid which is characterized by

enlargement of the thyroid gland and increased

hormone thyroid (Płoski, 2011;

Baratawijaya, 2012

and Tanwar, 2010).

The most common of GD treatment is an anti-

thyroid drug. The other treatments are radioactive

iodine or thyroidectomy. These therapies aim to treat

the symptoms caused by hyperthyroidism and

reducing the enlargement of the thyroid gland, but

all kinds of these therapies have side effects and the

possibility to show the symptoms of

hyperthyroidism again, called a relapse (Aggarwal,

2014). Anti-thyroid drugs are considered as the first-

line treatment since it is easily implemented, but it

takes 1-2 years to achieve remission and even 30-

50% of patients with GD take>2 Tahun (American

Thyroid Association, 2014). GD patients are

determined as remission when the levels of FT4,

FT3 and TSH in the serum are normal without

medication. In fact, more than 50 percent of patients

who had remission GD may experience repeated

symptoms of hyperthyroidism or may have relapse

(McKenna T Joseph, 2001 and Jacobson, et al.,

2007).

224

Amir, T., Suryandari, D., Eliana, F., Yunaini, L. and Yanti, D.

Association of sCD40 Level in Serum with Risk for Relapse in Graves’ Disease.

DOI: 10.5220/0009589402240227

In Proceedings of the 1st International Conference on Health (ICOH 2019), pages 224-227

ISBN: 978-989-758-454-1

GD is also known as a multifactorial disease,

which is this disease caused by environmental and

genetic factors. CD40 gene is one of the genes that

regulate immune responses on the follicular cells of

the thyroid gland (Huber, et al., 2012). CD40 signals

are required for immune response and pathogenesis

of the autoimmune disease (Peters, 2011). The

increase of CD40 expression leads to the formation

of antibodies that specific to the thyroid and produce

cytokines or chemokines in follicular cells thyroid

(Huber, et al., 2012).

Ligands of CD40 is CD40L/ CD154, expressed

on the surface of CD

T lymphocytes. The signal of

CD40-CD154 interaction indicates a significant role

to maintain the process of autoimmunity in the body

(Myśliwiec, 2007). Interaction between CD40 as a

costimulator molecules with its ligand CD154, is

necessary to activate T cells, B cells proliferation

and differentiation, production of immunoglobulin

(Ig), Ig class switching and also to induce the

proinflammatory cytokines, such as interleukin 6

(IL-6) (Van Kooten C, 2000 and Peters, et al., 2008).

Based on the previous research, showed that

sCD40 expression can affect etiology of GD.

Myśliwiec, et al., 2007 found that CD40-CD154

interaction has a role in the pathogenesis of Graves'

Ophthalmopathy (GO) and the serum level of CD40

(sCD40) had elevated. Other studies also

demonstrated a positive correlation between sCD40

with antibody to TSHR (aTSHR) in patients GD

(Mysliwiec, et al., 2007). These researchers

suggested that the presence of sCD40 in serum has a

role of the pathological disease. But until now, the

role of sCD40 influencing the physiological function

of CD40 for relapse in GD patients remains to be

studied more deeply. Therefore, further research is

needed to determine the role of sCD40 levels and the

risk for relapse as a marker of the active stage of

GD.

2 METHOD

2.1 Subjects

The selection of the case group (relapse) and control

group (remission) are based on the status of patients

in medical records.

2.2 Venous Blood Aspiration

A total of 2 mL venous blood sample was taken by

syringe and then put into a tube not containing

ethylenediaminetetraacetic acid (EDTA) to get a

sera. All the sera were kept frozen at -20°C until

used.

2.3 Measurement of sCD40 Level

sCD40 levels from all patients were performed using

a sandwich ELISA kits (Human sCD40 ELISA Kit

Platinum BMS 265, Bender Med Systems, Vienna,

Austria). This ELISA commercial kits were used to

determine the serum level of sCD40 concentration

from 7.8 to 1.000 pg/mL.

First, various

concentrations of standard solution was prepared by

dilution (500 pg/mL, 250 pg/mL, 125 pg/ml, 62.5

pg/ml, 31.3 pg/ml, 15.6 pg/mL and 7.8 pg/ m L).

Plate that has been coated with a specific

monoclonal antibody against of sCD40, wash with a

wash buffer (1x) twice (200 mL each well) and

allowed for 10-15 seconds. Next, 50 mL of each

standard solution and 50 mL sample test (serum)

were incorporated into different wells and then on

each well added 50 mL of Biotin-Conjugated

antibody (1x). Plate covered with adhesive film and

incubated for 2 hours at room temperature (18

C) on a shaker machine at 400 rpm. After that,

the plate was washed 10 times (each well 200 uL)

with wash buffer (1x), then added 100 mL

Streptavidin-Horseradish Peroxidase (HRP) on each

well. The plate was resealed with adhesive film and

incubated for 1 hour at room temperature (18

-25

on a shaker machine at 400 rpm. When the

incubation was completed, the plate is washed again

as much as 10 times (each well 200 uL) with wash

buffer (1x), then added 100 mL of Substrate

Solution (tetramethyl-benzidine) on each well and

incubated 10 min at room temperature (18

-25

C) in

darkroom (not exposed to direct light exposure).

Enzyme-substrate reaction was stopped by addition

of 100 mL of Stop Solution (1M phosphoric acid). A

colored product is formed in proportion to the

amount of human sCD40 present in the sample or

standard. Then the absorbance is measured with an

ELISA reader at 450 nm. A standard curve is

prepared from 7 human sCD40 standard dilutions

and human sCD40 sample concentration determined.

sCD40 concentration is determined by comparing

the OD value of the sample with a standard curve

(eBioscience).

2.4 Data Analysis

Independent t-test was used to show the association

between sCD40 levels with risk for relapse in GD

patients at p-value less than 0.05 was considered

significant.

Association of sCD40 Level in Serum with Risk for Relapse in Graves’ Disease

225

3 RESULT AND DISCUSSION

The normality test on each group showed that data

from relapse group has normal distribution (0.161),

but not in the remission group (<0.001). Then we did

the transformation data by log 10. The results were

showed normal distribution of data (relapse= 1.49

and remission= 0.079). Further, the data is tested by

an independent t-test. The results are presented in

Table 1.

Table 1: The association between sCD40 level in serum

with risk for relapse on GD patients.

Rela

se Remission

sCD40

level

(pg/mL)

1,62±0,17 1,46±0,14 <0,001

*independent t-test

Based on the results indicated that there is a

significant association between sCD40 levels with

risk for relapse on GD patients (p<0.001) at α=0.05.

GD patients who relapsed had an average serum

sCD40 levels higher than GD patients who did not

relapse.

In this study, the average levels of sCD40 in the

relapse group was higher than the remission group

and found a significant association between sCD40

level with risk for relapse in GD. This result

indicates that high levels of sCD40 in serum on GD

patients may increase the risk for relapse. A previous

study by Mysliwiec et al in Poland, also

demonstrated that sCD40 level was higher in GD

(hyperthyroidism) compared to patients with GD

(euthyroid), but did not show a significant

association (Mysliwiec, et al., 2007). Other studies

also measured the level of sCD40 in patients with

Graves' Ophthalmopathy (GO), GD

(hyperthyroidism), GD (euthyroid) and controls

(people who have no history of disease GO/GD),

showed that levels of sCD40 from high to low

concentration were patients with GO, GD

(hyperthyroidism), GD (euthyroid) and controls. The

statistical analysis showed a significantly different

levels of sCD40 only found on GO with control

patients (p<0.001), GO with GD (hyperthyroidism)

patients (p<0.001) and GD (hyperthyroidism) with

controls patients (p<0.05), but not seen a significant

difference between sCD40 level in GD

(hyperthyroidism) with GD (euthyroid) group

(Myśliwiec, et al., 2007).

The biological role of sCD40 in affecting

physiological function on GD patients is not clear.

Release of sCD40 is suspected from the

transmembrane receptor. It can occur because of

splicing while gene expression or proteolysis on the

transmembrane protein (Van Kooten C, 1996). High

levels of serum sCD40 in patients with relapsed,

indicates the increase of CD40 expression on

transmembrane region. Interaction of CD40, which

located on the membrane with CD40L will secrete

matrix metalloproteinase enzymes that act as a

protease to degradation of the protein (Karimi,

2012).

This condition caused by CD40 on

transmembrane region has proteolysis, with the

result that can be found in serum as a soluble form

(sCD40).

The higher sCD40 level on GD patients indicates

that sCD40 has contributed to development and risk

for relapse on GD, based on research conducted by

Mysliwiec et al that showed a positive correlation

between sCD40 with TSHR antibody (Mysliwiec, et

al., 2007). Other studies also showed that follicular

cell apoptosis decreased in patients with GD and

increased in HT (Kie JH, 2001).

The role of sCD40 is also suspected as an

inhibitor of the CD40-CD40L interaction, so that the

immune response decreases (Mysliwiec, et al.,

2007). This interaction is affected by the amount of

energy required for binding both sCD40 and CD40

that found on membrane with CD40L, to affect the

immune response. This hypothesis would be

stronger if the levels of CD40 on the membrane are

known, so it can be seen clearly differences of CD40

expression contained in the transmembrane or in the

form of dissolved (soluble) and its effects on the risk

of relapse.

Binding of CD40-CD40L occurs in the

multimerisasi form. Generally, the structure of

CD40 membrane is a trimerization form, while the

structure of sCD40 is not known with certainty yet.

As a result, interaction CD40-CD40L on the

membrane has a higher affinity than sCD40. Binding

of CD40 on the membrane with CD40L occurs

through binding in the form of dimerization or

trimerization, but trimerization will give a strong

response to intracellular signaling. CD40 and

CD40L have a trimerization form. So, this

interaction will create oligomerization which lead to

the introduction of protein adapters such as TRAFs

at the intracellular domain, improving signal cascade

and activates transcription factors that play a role in

producing proteins, which influence the immune

response in the body either humoral immune

responses or cellular and prevent apoptosis (Peters,

2011;

Ellmark, 2002

and Zazzeroni, et.al., 2001).

ICOH 2019 - 1st International Conference on Health

226

4 CONCLUSIONS

There was a significant association between sCD40

level in serum with risk for relapse on GD patients.

So, sCD40 levels in serum may be used as a marker

for the determination of GD activity.

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