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Evaluation of pentraxin-3 in familial Mediterranean fever patients during attack and attack-free periods

INTRODUCTION: Pentraxin-3 (PTX-3) is a prototype of pentraxin proteins that have been shown to be involved in acute phase response. In this study, we aimed to investigate the relationship between PTX-3 levels and familial Mediterranean fever (FMF) disease, and to evaluate PTX-3 as a novel diagnostic marker of FMF. METHOD: Forty-three male patients diagnosed with FMF and 42 healthy individuals were included in the study. Patients with other infl ammatory diseases and patients who used drugs having anti-infl ammatory properties were excluded from the research. Blood samples were obtained during both attack and attack-free periods. RESULTS: Patient attack periods were confi rmed by combining physical examination and elevation of acute phase reactants. Acute phase reactants were signifi cantly higher in attack versus attack-free periods (p < 0.01), however PTX-3 levels were not signifi cantly different between the two periods. Additionally, PTX-3 levels in FMF patients were higher than in controls in both attack (917.29 ± 725.29 vs 451.83 ± 291.95, p < 0.01) and attackfree periods (748.23 ± 487.53 vs 451.83 ± 291.95, p < 0.01). CONCLUSION: In this study, we showed that PTX-3 levels, in both FMF attack and attack-free periods, were signifi cantly higher than in the control group. Finally, PTX-3 may be a promising biomarker for FMF diagnosis and may predict FMF attacks (Tab. 2, Fig. 2, Ref. 18). Text in PDF www.elis.sk.


Introduction
Familial Mediterranean fever (FMF) is defi ned by repetitive periods of fever, infl ammation and pain in chest, abdomen or joints and with additional fi ndings such as myalgia and erysipelas-like skin lesions throughout life. Although FMF has a distinctly ethnic distribution, FMF patients have been reported in many countries all around the world. FMF is especially seen in the Mediterranean region in Sephardic Jews, Armenians, Turks, Greeks, Arabs and Italians (1)(2)(3)(4). It has been shown that there is a mutation in chromosome 16 in the Mediterranean fever (MEFV) gene (5). This gene encodes pyrin protein which has an important role in infl ammation and apoptosis (6). As a result of the MEFV mutation, pyrin and anti-infl ammatory protein production decreases and infl ammatory conditions cannot be suppressed, thereby resulting in fever and infl ammation at certain locations of the body.
Currently, there is no defi nitive technique for the diagnosis of FMF disease and the diagnosis is made by monitoring for clinical signs according to the criteria defi ned by Tel Hashomer (2)(3)(4). Although not necessary, detecting a mutation in MEFV (Mediterranean fever) gene favors the diagnosis in patients who have clinical fi ndings. The other fi nding that supports the diagnosis is that of elevated blood levels of acute phase reactants at the attack periods. It was found that there is an excessive activation of cytokine cascades during the disease (5)(6)(7)(8). Both attack-free and attack periods show a signifi cant increase in proinfl ammatory cytokines. Abnormal pyrin protein that occurs as a result of MEFV gene mutations makes the blocking suppression of infl ammation ineffective and is thought to be responsible for this ongoing infl ammatory process (9).
Pentraxin-3 (PTX-3) is an acute phase reactant located in the long pentraxin group. PTX-3 is produced as a result of infl ammatory cytokines such as TLR (toll-like receptor) agonists, IL-1β and tumor necrosis factor-alpha (TNF-α). There are abundant cells that play a role in PTX-3 production, such as dendritic cells, monocytes, macrophages, vascular endothelial cells, kidney epithelial cells, fi broblasts, adipocytes, smooth muscle cells, glial cells, mesangial cells and synovial cells (10). Infl ammation has been shown to be associated with increased levels of serum PTX-3 in several studies (11)(12)(13). Our aim was to investigate the levels of PTX-3 among FMF patients, and to evaluate PTX-3 as a novel diagnostic marker of FMF.

Materials and methods
In this study, 43 male patients who were admitted to our department and diagnosed with FMF between November 2013 and March 2014 were accepted as the patient group and 42 healthy male individuals who had similar characteristics and did not have any active or chronic disease were accepted as the control group. This study was authorized by our hospital's medical ethics committee. Informed consents were signed by all subjects in this study.
FMF attack period was diagnosed by combining a positive physical examination with high levels of acute phase reactants (APR). All pain locations were examined and noted for further evaluation and classifi cation. During both FMF attack-free and attack periods, physical examination was performed, and blood samples were taken in order to evaluate APR levels (erythrocyte sedimentation rate (ESR), fi brinogen and leucocyte count). Blood samples were obtained in the fi rst 24 hours of the attack period, and at least 10 days after the last FMF attack in an attack-free period. Patients who used nonsteroidal anti-infl ammatory drugs, steroids, immune suppressed or immune-modulating drugs and patients who had chronic diseases such as cardiovascular disease, acute or chronic liver or kidney disease, chronic obstructive lung disease, rheumatologic disease, malignancy, thyroid disease, immune defi ciency, hypertension, diabetes mellitus, acute/chronic infection, bleeding disease and those who did not completely fulfi ll the criteria for research were excluded from the study.
Blood samples were centrifuged for 5 minutes at 5000 rpm and a volume of 2 cc of serum was separated into Eppendorf tubes for PTX-3 studies and stored at −80 °C. After reaching a suffi cient sample size, all samples were analyzed in the biochemistry lab.
ESR was examined with a Vacuet at Sed Rate Screener 100 device by precipitation, fi brinogen was examined with an Architech ci8200 device, and complete blood count was examined with a Cell-Dyn Sapphire device. PTX-3 was examined with a Booster Elx800 Instruments-Biotek Biotek Plate Reader and Elpan-40 by ELISA using an Instruments-Plate Washer.

Statistical analysis
The research was designed as an observational, prospective, case-control study. SPSS 15.0 was used for statistical analysis. The average value of the parameters in the patient and control groups, range of values and standard deviations were calculated, and Kolmogorov-Smirnov test was used for compliance of the data with normal distribution. Student T-test and Mann-Whitney U test were used for comparisons. Roc analysis was used to evaluate the sensitivity and specifi city for PTX-3 in patients with FMF. Pearson analysis was used for correlations. p < 0.05 was assessed as statistically signifi cant.

Results
Forty-three male FMF patients with attack periods and 42 healthy controls were included in the study. The control group consisted of healthy volunteers of similar age and gender. The data of all groups are shown in Table 1.
When data from patients with FMF attacks and asymptomatic periods were compared, APRs were higher in the attack period (p < 0.01) (Tab. 1). PTX-3 levels in the attack group were higher than in the asymptomatic period, but this difference was not statistically signifi cant (p > 0.05).
White blood cell, ESR, fi brinogen and PTX-3 levels in the attack period in FMF patients were signifi cantly higher than in the control group (Fig. 1).
Although ESR and fi brinogen were within normal values, they were signifi cantly higher in the attack-free group compared to the control group. Additionally, PTX-3 levels were signifi cantly higher in the asymptomatic period (748.23 ± 487.53 vs 451.83 ± 291.95; p < 0.01). No differences were found in terms of age and white blood cell count (p>0.05).

Tab. 1. Data of patient groups. Reactant levels in attack period were signifi cantly higher than in both attack-free period and control group.
Pentraxin-3 (PTX-3) level in attack period was signifi cantly higher when compared to that in control group, but not signifi cantly higher when compared to that in asymptomatic period.

Fig. 1. Comparison of PTX 3 levels between FMF patients and healthy controls according to attack period. The PTX 3 levels in both attack
period and attack free period.were signifi cantly higher than in control group.
The sensitivity, specifi city, positive and negative predictive values of PTX-3 in acute attack and asymptomatic periods are shown in Table 2, and ROC curves of attack and asymptomatic periods are shown in Figure 2. When a PTX-3 level cut-off value of 696.2 was used, the test's positive predictive value was 85.2 % with 71.6 % overall accuracy (Tab. 2). According to correlation analysis, there was no correlation between white blood cell count, fi brinogen, ESR and PTX.

Discussion
In this study, we evaluated the association between PTX-3 levels and other infl ammation markers in FMF patients during both symptomatic and asymptomatic periods. We showed that PTX-3 levels were signifi cantly higher in FMF patients than in healthy individuals, regardless of attack period.
During the attack period in FMF disease, higher levels of ESR, fi brinogen and white blood cell count when compared to an attackfree period is an expected result and has been shown in many studies (10,14). Similarly, we found elevated APRs in the FMF attack period. As opposed to these studies, we found ESR, fi brinogen and leukocyte levels to be signifi cantly higher in the asymptomatic period than in the control group. Nevertheless, these parameters were also within normal levels in the asymptomatic period. Therefore, we think that this signifi cance might not be important. In addition to these fi ndings, Bulut et al (14) demonstrated elevated PTX-3 levels in FMF patients when compared with healthy controls. We found similar fi ndings in our study. Interestingly, PTX-3 levels were also higher in asymptomatic period when compared with the control group, while this elevation in attack period did not differ from that in attack-free period. Therefore, these fi ndings may be a clue to PTX-3 as a diagnostic laboratory test for suspected FMF.
PTX-3 is one of the major acute phase reactants, and in infl ammatory conditions serum levels can increase up to 3-5 times from the basal value. PTX-3 is produced as a result of infl ammatory cytokines such as TLR (toll-like receptor), IL-1β and TNF-α. PTX-3 is stored in specifi c granules in neutrophils and is released into the blood as a result of stimulation of TLRs. In addition, IL-1β and IL-6 stimulate the release of large amounts of PTX-3 from peripheral mononuclear cells (11). PTX-3 -pentraxin-3; PPV -positive predictive value; NPV -negative predictive value

a b
Studies of PTX-3 pointed out that it increases in some infl ammatory diseases (12,13). Ishihara et al (12) reported that serum PTX-3 levels were higher in the period of active disease of Takayasu arthritis when compared to both inactive period and a control group. Finally, they concluded that PTX-3 is more specifi c than CRP in demonstrating arterial infl ammation. In a study investigating PTX-3 levels with forty-three patients with vasculitis, PTX-3 levels were higher in active vasculitis than in inactive period of vasculitis (13). In addition, PTX-3 levels were similar to those in healthy control group and inactive vasculitis group. As opposed to the latter fi ndings and in accordance with Bulut et al (14), we found that serum PTX-3 levels were signifi cantly higher in the asymptomatic period. This difference from other infl ammatory conditions might be described as an ongoing subclinical infl ammation in FMF disease. Thus, our fi ndings may suggest that PTX-3 levels may be used as a diagnostic marker for FMF disease.
In this study, PTX-3 levels were absolutely higher in the attack period than in the attack-free period, but this result was not signifi cant. First of all, colchicine treatment may cause this consequence since colchicine has anti-infl ammatory effects and can cause a decrease in proinfl ammatory cytokine levels (15)(16)(17). Another reason may be in the cross-sectional design of the study and the results of samples refl ect just one time point. PTX-3 serum levels do not have to reach peak levels at the time of blood test. Therefore, studies with a larger number of patients and with blood samples taken consecutively for 2 or 3 days of attack for PTX-3 measurement may give more accurate results.
One of the important results of our study was that PTX-3 levels were higher in attack or attack-free patients than in the healthy individuals. Studies have shown that also in attack-free periods, infl ammation continues. Duzova et al (16) showed that in the attackfree period, subclinical infl ammation continued in FMF patients and they stated that SAA may be a good marker for demonstrating subclinical infl ammation. Haznedaroglu et al (18) showed that cytokines IL-17 and IL-18 remained high in the attack-free periods. Therefore, as with other acute phase reactants, high levels of PTX-3 may indicate that PTX-3 has a role in the pathogenesis of subclinical infl ammation in FMF patients, and that PTX-3 may be used as a biomarker for the determination of infl ammatory process in FMF.
Our study has some limitations. Firstly, the study population was not appreciably large. However, FMF is a rare disease, and it is not easy to include FMF patients in both periods. Secondly, all study participants were male, since the research was carried out in a tertiary military center.
In conclusion, we have shown that APRs were signifi cantly higher in the attack period in accordance with current literature and that PTX-3 levels were signifi cantly higher in FMF patients than in healthy controls regardless of the disease period. Thus, this difference may help in the diagnosis of FMF disease regardless of the attack period of the disease.