ABSTRACT

Objective:

Cystic echinococcosis (CE) is one of the most common zoonotic diseases worldwide. Diagnosis of CE is predominantly based on imaging techniques and serological tests are used in cases of non-characteristic imaging findings as diagnostic reference. However, serological test results cannot be completely reliable as they are affected by multi-factors. P-selectin and resistin are inflammatory markers that are altered during the acute stages of infection. In this purpose, inflammatory markers as P-selectin and resistin have been investigated for a potential diagnostic reference for CE diagnosis.

Methods:

A total of 60 patients who were diagnosed with CE and twenty-five healthy individuals were included in this study. Blood samples were obtained from all participants. Obtained sera were evaluated using the P-selectin and resistin ELISA kits for protein levels. Additionally, the relative expression of SELP (P-selectin) and RETN (resistin) genes were determined using the comparative CT (ΔΔCT) method between groups as CE patients with active and inactive cysts, CE patients and healthy controls.

Results:

SELP (13.9-fold change, p<0.05) and RETN (8.1-fold change, p<0.05) were differentially expressed in CE patients compared in the control group. Whereas resistin protein levels were significantly higher in CE patients than the healthy controls (p<0.001), the difference in P-selectin protein levels was not significant (p>0.05). There was no difference between active and inactive CE patients in terms of P-selectin and resistin in gene and protein levels (p>0.05).

Conclusion:

Although there was no difference between the active and inactive CE patients, the good differentiation between the healthy controls and the CE patients suggested that resistin is a potential inflammatory diagnostic reference.

Keywords: Cystic echinococcosis, resistin, P-selectin, inflammatory marker, hydatid cyst

INTRODUCTION

Cystic Echinococcosis (CE) is one of the neglected diseases that affects around one million people worldwide. The causative agent of CE is larval form of Echinococcus granulosus sensu lato (s.l) (1). The infection always begins asymptomatically and clinical symptoms can vary from mild to severe. Although the liver is the most commonly affected organ for CE, it can be involved in every organ (2,3). Non-invasive imaging techniques especially ultrasonography (US) are the main tool for the diagnosis of CE. According to the World Health Organization-Informal Working Group on Echinococcosis (WHO-IWGE) classification, three clinical group have been identified as active (CE1 and CE2), transitional (CE3a/CE3b) and inactive (CE4 and CE5) (4). Until recently, surgery has been considered the only definitive therapy for liver CE, but new anthelmintic drugs and percutaneous interventional methods have extended the range of CE treatment. Even though there are a lot of options for treatment of CE, according to WHO-IWGE, the stage-specific approach should be implemented (2,3,5,6). For diagnosis, serological tests are used as supportive, however, the results can be deceptive due to variable sensitivity and specificity values (7). In addition, serodiagnosis can remain positive for years even after successful treatment (8). Therefore, serodiagnosis is not suitable to use for screening and confirm the presence of active infection.

Inflammatory markers can be used as biomarkers of active infection particularly for asymptomatic diseases (9). During inflammation, P-selectin (CD62P) is known as a mediator of leukocyte recruitment (10). P-selectins have a critical role in the parasite-induced (parasites of Schistosoma genus) progression of chronic liver disease (9). Resistin is a member of the resistin-like molecule (RELM) family of cysteine-rich secreted proteins that are present in humans (resistin and RELMb) and mice (11). Resistin is showed hormone-like activity at the beginning of inflammatory processes (12,13). Certain murine RELM proteins (RELMa and RELMb) are influenced by helminth infection (14). This study aims to determine protein and gene levels of P-selectin and resistin and to evaluate the potential of these inflammatory markers to be a diagnostic reference in the serum of CE patients.

METHODS

Ethical Considerations

The research protocol followed ethical guidelines of the Declaration of Helsinki. An informed consent that explained the purposes, benefits, and risks of the study was signed by the participants. This study was evaluated and accepted by the Hacettepe University Institutional Ethical Committee (no: 2020/16-72).

Sample Collection

A total 60 patients who were diagnosed with CE aged between 18-90 years after abdomen US examination were included in this study. All CE cysts were classified using the WHO-IWGE classification. In addition, twenty-five healthy individuals were included in this study after the US examination. Blood samples were taken from patients at the first diagnosis.

Serological Tests

All the collected sera were stored at -80 °C until study. The obtained serum samples were evaluated using the commercial serological test [Human P-selectin ELISA Kit (BT LAB, China) and Human Resistin ELISA Kit (BT LAB, China)] according to the manufacturer’s instruction. All tests were performed in a same session.

RNA Extraction, cDNA Synthesis and Real-time-Polymerase Chain Reaction (RT-PCR)

Total RNA extraction was performed from serum of the patients and healthy controls using RNA Extracol (EURx, Poland) following the manufacturer’s instructions. The RNA was quantified by FLUOstar Omega Microplate Reader (BMG LABTECH, Germany) using LVis plate. For cDNA synthesis, smART First Strand cDNA Synthesis kit (EURx, Poland) was used according to manufacturer’s recommendations.

Expression levels of SELP (P-selectin) and RETN (resistin) genes were determined in the serum of CE patients and healthy controls. The primers for SELP and RETN genes were selected according to previous studies (15,16). RT-PCR were performed using SYBR Green Master Mix (A.B.T., Turkey) and primers at 200 nM final concentration with ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific). The relative expression of SELP and RETN genes were determined using comparative CT (∆∆CT) method between CE patients and healthy controls. For normalization, GAPDH gene was used.

Statistical Analysis

GraphPad Prism 8.4.3 software was used for the statistical analyses. The results were interpreted with Mann-Whitney U test. The p-value under 0.05 was accepted as statistical significance.

RESULTS

Characteristics of Patients

Majority of the patients were female (63.3%, 38/60). The age range of the patients was between 18-85 years, and the mean age was 39.3. According to the WHO-IWGE classification system, 24 out of 80 CE cysts were identified as CE1, 14 as CE2, 3 as CE3a, 6 as CE3b, 28 as CE4 and 5 as CE5 respectively. Most of the patients had a single cyst (47/60, 78.3%), the rest harbored multiple CE cysts. No patient had a different type of hydatid cysts together. The average cyst diameter was recorded as 7.2 cm. The great majority of the CE cysts were involved in the liver (72/80, 90%), and also other organ involvements [spleen (2/80, 2.5%), kidney (3/80, 3.75%), bone (2/80, 2.5%) and intramuscular (1/80, 1.25%)] were recorded. All the patient’s characteristics are presented in Table 1.

Table 1

Expression Levels of SELP and RETN Genes

Comparative analysis was performed between 25 healthy control and 60 CE patients for the expression levels of SELP and RETN genes using RT-PCR. SELP (13.9-fold change, p<0.05) and RETN (8.1-fold change, p<0.05) were differentially expressed in CE patients compared to control group (Figure 1). Additionally, SELP and RETN gene expressions were compared in patients with active (38/60) and inactive (22/60) cysts and there was no significant difference (Figure 2).

Figure 1

Figure 2

Levels of Protein Concentration

The levels of P-selectin and Resistin for CE patients and healthy controls were detected using ELISA and presented in Figure 3. Whereas resistin levels were significantly higher in CE patients than the healthy controls (p<0.001), the difference in P-selectin levels was not significant (p>0.05).

Figure 3

DISCUSSION

According to the WHO data, more than one million people are living with CE and alveolar echinococcosis (AE) worldwide at any one time and incidence data of human CE does not reflect real prevalence value due to the asymptomatic nature of the infection (17). According to the data of the Ministry of Health, while the morbidity rate was reported as 0.57 per 100,000 in 2008, it was reported as 2.25 in 2019 in Turkey (18). The most valuable epidemiological data for CE are obtained with active surveillance methods and screening using USG, unfortunately, these are both labor-intensive and costly. Hence, there is a need for new molecules that can be supportive markers for diagnosis.

Inflammatory markers have been studied in various diseases including helminth infections. Until now, several ex vivo studies on CE have demonstrated that the association between cytokine levels and disease progression (19,20). According to a previous report, compared to healthy individuals, levels of Interferon- (IFN-), Interleukin-12 (IL-12), IL-16, IL-18, IL-4, IL-5, IL-10, and IL-13 were found to be higher in the serum of CE patients (21). Among the inflammatory markers, P-selectin and resistin has been evaluated in some parasitic infections (9). However, there is lack of data on their role in CE. In this study, P-selectin and resistin expression were investigated at both gene and protein levels in CE patients compared with healthy controls.

P-selectin (CD62P) is among the immune system components and can play a role as a mediator in leukocyte recruitment during inflammation (22). Besides, P-selectin is an adhesion molecule that migrates to the surface of endothelial cells and platelets under inflammatory stimuli (23). Platelets are among the immune system components and can play a role as mediator cells in inflammation via potent pro-inflammatory substances (24,25). Platelets are involved in the protection against helminths as potent effector cells (26). A previous report showed that CD62P expression was significantly upregulated in CE patients compared to the control group, suggesting the presence of platelet activation during CE infection (27).

In this study, we have confirmed that SELP gene expression (13.9-fold change, p<0.05) were found to be significantly up-regulated in the CE patients compared to healthy controls. In other studies, it is reported that the correlation between gene expression measured at the mRNA level and the corresponding protein level is generally weak (28-30). Thus, protein levels of P-selectin were also investigated to observe the correlation between the mRNA expressions and protein levels. However, there was no statistically significant difference in protein levels of P-selectin between CE patients and healthy controls.

On the other hand, biological effects of human resistin have been investigated in helminth and viral infections though its exact function in humans is still unclear. Based on previous studies, plasma resistin levels were up-regulated in both chronic filarial nematode infection and soil-transmitted helminth infection (14). In addition, it was found to be correlated with increased parasite burden and elevated levels of inflammatory cytokines TNF, CCL2 and IL-6 (31). According to our results, compared to healthy controls relative RETN expression was significantly higher in CE patients. Besides, protein levels of resistin were significantly elevated in CE patients, as well. Previous reports indicated that resistin expression was increased in macrophages from patients with filarial infection following exposure to parasite antigen (32,33). In addition, a lot of studies have demonstrated that lipopolysaccharide (component of Gram-negative bacteria) can influence resistin expression in vivo and in vitro. However, accumulating data has shown that resistin expression is a part of innate immune response to various helminth infection. Hence, resistin may play different roles in two types of immune responses as type 1 inflammatory process triggered by bacterial components and Th2 based immune response induced by helminths (14).

We have also performed the comparison of P-selectin and resistin expression levels in patients with active and inactive cysts and found no difference between the two groups of CE patients. Although there was no difference between the active and inactive patients, the good differentiation between the healthy controls and the CE patients suggested that they may be potential inflammatory diagnostic references.

CONCLUSION

P-selectin and resistin serum levels have been comparatively evaluated between CE patients and healthy controls for the first time. In addition, the present study has demonstrated that resistin is a potential inflammatory marker for CE diagnosis. More investigations characterizing the infection on the inflammatory pathways should be conducted, especially for the asymptomatic people with poor access to healthcare groups.

*Ethics

Ethics Committee Approval: The research protocol followed ethical guidelines of the Declaration of Helsinki. This study was evaluated and accepted by the Hacettepe University Institutional Ethical Committee (no: 2020/16-72).

Informed Consent: Informed consent that explained the purposes, benefits, and risks of the study was signed by the participants.

Peer-review: Internally and externally peer-reviewed.

*Authorship Contributions

Surgical and Medical Practices: T.Ç., E.Ü., A.B.D., D.A., O.A., Concept: S.Ö., T.Ç., E.Ü., A.B.D., D.A., Y.A., O.A., Design: S.Ö., S.Y.Ç., Y.A., O.A., Data Collection or Processing: S.Ö., İ.B., Analysis or Interpretation: S.Ö., İ.B., S.Y.Ç., Literature Search: S.Ö., İ.B., Writing: S.Ö., İ.B., S.Y.Ç., Y.A., O.A.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

References

Tamarozzi F, Akhan O, Cretu CM, Vutova K, Akinci D, Chipeva R, et al. Prevalence of abdominal cystic echinococcosis in rural Bulgaria, Romania, and Turkey: a cross-sectional, ultrasound-based, population study from the HERACLES project. Lancet Infect Dis 2018; 18: 769-78.

Akhan O, Ozmen MN, Dinçer A, Sayek I, Göçmen A. Liver hydatid disease: long-term results of percutaneous treatment. Radiology 1996; 198: 259-64.

Brunetti E, Kern P, Vuitton DA. Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. Acta Trop 2010; 114: 1-16.

Brunetti E, Tamarozzi F, Macpherson C, Filice C, Piontek MS, Kabaalioglu A, et al. Ultrasound and Cystic Echinococcosis. Ultrasound Int Open 2018; 4: E70-8.

Akhan O, Erdoğan E, Ciftci TT, Unal E, Karaağaoğlu E, Akinci D. Comparison of the Long-Term Results of Puncture, Aspiration, Injection and Re-aspiration (PAIR) and Catheterization Techniques for the Percutaneous Treatment of CE1 and CE3a Liver Hydatid Cysts: A Prospective Randomized Trial. Cardiovasc Intervent Radiol 2020; 43: 1034-40.

Akhan O, Salik AE, Ciftci T, Akinci D, Islim F, Akpinar B. Comparison of Long-Term Results of Percutaneous Treatment Techniques for Hepatic Cystic Echinococcosis Types 2 and 3b. AJR Am J Roentgenol 2017; 208: 878-84.

Menezes da Silva A. Hydatid cyst of the liver-criteria for the selection of appropriate treatment. Acta Trop 2003; 85: 237-42.

Pagnozzi D, Addis MF, Biosa G, Roggio AM, Tedde V, Mariconti M, et al. Diagnostic Accuracy of Antigen 5-Based ELISAs for Human Cystic Echinococcosis. PLoS Negl Trop Dis 2016; 10: e0004585.

Chimponda TN, Mushayi C, Osakunor DNM, Vengesai A, Enwono E, Amanfo S, et al. Elevation of C-reactive protein, P-selectin and Resistin as potential inflammatory biomarkers of urogenital Schistosomiasis exposure in preschool children. BMC Infect Dis 2019; 19: 1071.

Kamel MM, Fouad SA, Basyoni MM. P selectins and immunological profiles in HCV and Schistosoma mansoni induced chronic liver disease. BMC Gastroenterol 2014; 14: 132.

Yang RZ, Huang Q, Xu A, McLenithan JC, Eisen JA, Shuldiner AR, et al. Comparative studies of resistin expression and phylogenomics in human and mouse. Biochem Biophys Res Commun 2003; 310: 927-35.

Patel SD, Rajala MW, Rossetti L, Scherer PE, Shapiro L. Disulfide-dependent multimeric assembly of resistin family hormones. Science 2004; 304: 1154-8.

Holcomb IN, Kabakoff RC, Chan B, Baker TW, Gurney A, Henzel W, et al. FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family. EMBO J 2000; 19: 4046-55.

Jang JC, Chen G, Wang SH, Barnes MA, Chung JI, Camberis M, et al. Macrophage-derived human resistin is induced in multiple helminth infections and promotes inflammatory monocytes and increased parasite burden. PLoS Pathog 2015; 11: e1004579.

Janke J, Engeli S, Gorzelniak K, Luft FC, Sharma AM. Resistin gene expression in human adipocytes is not related to insulin resistance. Obes Res 2002; 10: 1-5.

Gillies PJ, Richardson NA, Walshe J, Stephenson SA, Dawson RA, Harkin DG. Demonstration of P-selectin expression and potential function in human corneal epithelial cells. Exp Eye Res 2018; 176: 196-206.

Ok ÜZ, Kilimcioğlu AA, Özkol M. [Cystic Echinococcosis in Humans in Turkey]. Mikrobiyol Bul 2020; 54: 510-22.

Topluoglu S. Current Situation Report of Cystic Echinococcosis in Turkey. Turk Hij Den Biyol Derg 2020; 77: 1-52.

Riganò R, Profumo E, Ioppolo S, Notargiacomo S, Teggi A, Siracusano A. Serum cytokine detection in the clinical follow up of patients with cystic echinococcosis. Clin Exp Immunol 1999; 115: 503-7.

Bayraktar MR, Mehmet N, Durmaz R. Th1 and Th2 inducing cytokines in Cystic echinococcosis. Turkiye Parazitol Derg 2005; 29: 167-70.

Mezioug D, Touil-Boukoffa C. [Cytokine profile in human hydatidosis: possible role in the immunosurveillance of patients infected with Echinococcus granulosus]. Parasite 2009; 16: 57-64.

Ley K. The role of selectins in inflammation and disease. Trends Mol Med 2003; 9: 263-8.

Perkins LA, Anderson CJ, Novelli EM. Targeting P-Selectin Adhesion Molecule in Molecular Imaging: P-Selectin Expression as a Valuable Imaging Biomarker of Inflammation in Cardiovascular Disease. J Nucl Med 2019; 60: 1691-7.

Klinger MH. Platelets and inflammation. Anat Embryol 1997; 196: 1-11.

Küçükbayrak A, Oz G, Fındık G, Karaoğlanoğlu N, Kaya S, Taştepe I, et al. Evaluation of platelet parameters in patients with pulmonary hydatid cyst. Mediterr J Hematol Infect Dis 2010; 2: e2010006.

Bout D, Joseph M, Pontet M, Vorng H, Deslée D, Capron A. Rat resistance to schistosomiasis: platelet-mediated cytotoxicity induced by C-reactive protein. Science 1986; 231: 153-6.

Matowicka-Karna J, Kemona H, Dymicka-Piekarska V, Butkiewicz A. Activation of blood platelets in echinococcosis--CD62P and CD63 expression. Parasitol Res 2006; 98: 214-7.

Gry M, Rimini R, Strömberg S, Asplund A, Pontén F, Uhlén M, et al. Correlations between RNA and protein expression profiles in 23 human cell lines. BMC Genomics 2009; 10: 365.

Maier T, Güell M, Serrano L. Correlation of mRNA and protein in complex biological samples. FEBS Letters 2009; 583: 3966-73.

Chen G, Gharib TG, Huang CC, Taylor JM, Misek DE, Kardia SL, et al. Discordant protein and mRNA expression in lung adenocarcinomas. Mol Cell Proteomics 2002; 1: 304-13.

Pine GM, Batugedara HM, Nair MG. Here, there and everywhere: Resistin-like molecules in infection, inflammation, and metabolic disorders. Cytokine 2018; 110: 442-51.

Babu S, Kumaraswami V, Nutman TB. Alternatively activated and immunoregulatory monocytes in human filarial infections. J Infect Dis 2009; 199: 1827-37.

Semnani RT, Mahapatra L, Moore V, Sanprasert V, Nutman TB. Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi. Infect Immun 2011; 79: 3957-65.

Evaluation of Potential Inflammatory Markers for Cystic Echinococcosis: P-selectin and Resistin