• Users Online: 77
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2023  |  Volume : 6  |  Issue : 1  |  Page : 32-37

The relationship between coronavirus disease-2019-positive patients and plasma interleukins and transforming growth factor-β levels


1 Department of Histology and Embryology, Faculty of Medicine, Manisa Celal Bayar University, Manisa, Turkey; DESAM Institute, Near East University, Nicosia, North Cyprus
2 DESAM Institute, Near East University, Nicosia, North Cyprus; PCR Unit, Clinical Laboratory, Faculty of Medicine, Kocaeli University, Mugla, Turkey
3 Department of Histology and Embryology, Faculty of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
4 Department of Clinical Microbiology and Infection Diseases, Faculty of Medicine, Kocaeli University, Mugla, Turkey
5 Derince Training and Research Hospital, Health Science University, Kocaeli, Mugla, Turkey
6 DESAM Institute, Near East University, Nicosia, North Cyprus; Department of Clinical Microbiology, Faculty of Medicine, Manisa Celal Bayar University, Manisa, Turkey

Date of Submission30-Mar-2022
Date of Decision30-Aug-2022
Date of Acceptance11-Sep-2022
Date of Web Publication3-Jan-2023

Correspondence Address:
Melike Ozgul-Onal
Department of Histology and Embryology, Faculty of Medicine, Mugla Sitki Kocman University, Mugla
Turkey
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsm.jnsm_37_22

Rights and Permissions
  Abstract 


Objective: The aim of this study is to reveal the relationship between the cytokine plasma levels and symptoms of coronavirus disease-2019 (COVID-19)-positive patients, which is characterized by serious respiratory syndromes. Materials and Methods: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2)-positive patients were evaluated in four groups. Group 1 patients had no symptoms. Group 2 patients were mildly symptomatic. Group 3 patients had multiple symptoms. Group 4 patients had all symptoms of acute respiratory distress syndrome. Analysis of interleukin (IL)-17A, transforming growth factor-β1 (TGF-β1), and IL-6 concentrations in plasma samples taken from patients were examined by enzyme-linked immunosorbent assay method. Results: IL-17A levels were increased in parallel with the clinical condition in all patients. TGF-β1 was only observed in patients in Groups 3 and 4, and IL-6 was only observed in Group 4 patients. Conclusion: It is known that many cytokines are involved in the development of different viral infections and viral invasion always triggers an inflammatory response. The profile of inflammatory markers may be used to classify COVID-19 patients. In conclusion of this study, it is suggested that the level of cytokines which is changed according to the patient's clinical status should be used to evaluate the response of SARS-CoV-2 treatment. IL-17A, TGF-β1, and IL-6 concentrations in plasma levels could be good prognostic indicators of COVID-19.

Keywords: Coronavirus disease-2019, cytokine, enzyme-linked immunosorbent assay, interleukin, plasma


How to cite this article:
Vatansever H S, Sayan M, Ozgul-Onal M, Akhan S, Ozel S, Sanlidag T. The relationship between coronavirus disease-2019-positive patients and plasma interleukins and transforming growth factor-β levels. J Nat Sci Med 2023;6:32-7

How to cite this URL:
Vatansever H S, Sayan M, Ozgul-Onal M, Akhan S, Ozel S, Sanlidag T. The relationship between coronavirus disease-2019-positive patients and plasma interleukins and transforming growth factor-β levels. J Nat Sci Med [serial online] 2023 [cited 2023 Jan 28];6:32-7. Available from: https://www.jnsmonline.org/text.asp?2023/6/1/32/366998




  Introduction Top


Coronaviruses which are named for the spikes that protrude from their surfaces are a family of hundreds of viruses and they can cause illnesses of the respiratory and enteric tract in both animals and humans.[1] In 2020, the new virus has been named severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the disease was called coronaviruses disease-2019 (COVID-19) which resulted in a global pandemic.[1] This outbreak has caused many clinical, economical, and physiological collapses worldwide. SARS-CoV-2 has high virulence with an incubation period of 2–14 days and it is transmitted by breathing or by contact with infected droplets. The common symptoms of COVID-19 are fever (85%–90%), myalgia (10%–15%), cough (65%–70%), headache (10%–15%), nausea/vomiting/diarrhea (<5%), and dyspnea (15%–20%). The symptomatic levels range from no symptoms in 30%, mild/moderate symptoms in 55%, and severe symptoms in 10% to critical symptoms in 5% of cases.[2] In parallel with this rapid spread occurring worldwide, the discovery of the molecular mechanisms triggered by the disease is necessary to reveal novel treatment strategies.[3]

During viral infection and some immunomodulatory molecules called cytokines are produced to support immune response. Interleukin-17A (IL-17A) is a major cytokine that regulates different immune functions such as the expression of other cytokines and chemokines, activation of leukocytes, and secretion of antibodies. In the host response to viral infections, the role of IL-17A is not clear. However, some studies suggest that IL-17A may protect individuals from some viral infections.[4] On the other hand, it was reported that IL-17 inhibits apoptosis and supports virus survival. It has the ability to block the Fas–FasL pathway in infected cells. The synergistic behavior between IL-6 and IL-17 was shown in Theiler's murine encephalomyelitis virus and it was concluded that the inhibition of apoptosis occurs in infected cells through induction of Bcl-2 and Bcl-xL (antiapoptotic proteins) by these IL.[5]

Transforming growth factor-β (TGF-β) is a pleiotropic cytokine which controls different immune responses. In viral infections such as HIV, HBV, or HCV, TGF-β levels in serum or in infected cells were reported to be increased in patients and correlated with disease progression.[6] The TGF-β signaling pathway and TGF-β protein expression or activation can be modulated by viral pathogens and other viruses. It is clearly known that when TGF-β1 is activated, cell proliferation, differentiation, apoptosis, epithelial–mesenchymal transition, and migration could be triggered in normal development and diseases.[7]

IL-6 is an important cytokine that is secreted from various cell types under the effect of viral infections. When IL-6 reaches its specific receptor, the JAK/STAT signaling pathway mostly becomes activated. IL-6 has two-edged behaviors as pro-and anti-inflammatory effects. In different virus models or during different viral infections, it was shown by several studies that upregulation of IL-6 may promote virus survival.[8]

It is known that many cytokines are involved in the development of different viral infections and viral invasion always triggers an inflammatory response. The profile of inflammatory markers may be used to classify COVID-19 patients. The aim of this study is to reveal the relationship between the cytokine plasma level changes of COVID-19 patients with different symptoms. This study focuses on obtaining results that can provide preliminary data in the choice of treatment by evaluating the relationship between plasma cytokine levels and different symptoms in COVID-19-positive patients in Turkey.


  Materials and Methods Top


Ethical statement

The ethical permission was approved by the Ethics Committee of Near East University (Protocol No: YDU/2020/78-1053) on April 23, 2020. Furthermore, this project was approved by the Republic of Turkey Ministry of Health (no: 2020-06-01T11_12_33). The samples were collected in June 2020 in Kocaeli, Turkey. Written informed consent was obtained before the study and the procedures were performed adhered to the ethical guidelines of the Declaration of Helsinki. All methods were performed in accordance with the relevant guidelines and regulations.

Study design, patient selection, and experimental groups

The plasma samples were obtained from the Kocaeli University Research and Application Hospital, Health Sciences University Kocaeli Derince Training and Research Hospital, and Kocaeli Darica Farabi Training and Research Hospital. Oro/nasopharyngeal swab samples from patients were sent for SARS-CoV-2 diagnosis to the routine polymerase chain reaction (PCR) unit of Kocaeli University and COVID-19-positive patients were identified using the reverse transcription-PCR method.[9]

The study was evaluated based on four different groups of COVID-19-positive patients depending on their symptomatic levels. Group 1 patients (n = 5) had no symptoms. Group 2 patients (n = 8) were moderately symptomatic (at least one of the symptoms of fever, headache, myalgia, nausea, vomiting, diarrhea, bone pain, sore throat, or shortness of breath) who had started treatment or not. Group 3 patients (n = 8) had multiple symptoms and had been hospitalized due to fever, headache, myalgia, nausea, vomiting, diarrhea, bone pain, sore throat, or shortness of breath and had started treatment. Group 4 patients (n = 9) had all symptoms of acute respiratory distress syndrome (ARDS) and had been hospitalized due to fever, cough, dyspnea, tachypnea, hypoxemia, hypotension, widespread radiological findings, and changes in consciousness [Table 1].
Table 1: Experimental groups of the study

Click here to view


Enzyme-linked immunosorbent assay

]TGF-β1 (Catalog No: EH0287) and IL-6 (Catalog No: EH0201) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Fine Test (China). The IL-17A (Catalog No: BMS2017) ELISA kit was purchased from Invitrogen by Thermo Fisher Scientific (Austria). The plasma samples of COVID-19-positive patients were centrifuged at 1000 × g 4°C for 20 min. The pellet was removed and 50 μl/well of supernatant was used. ELISA assays were performed and the solutions were prepared according to the manufacturer's instructions. The absorbance of the wells was read at the manufacturer's recommended wavelength (450 nm) using a microplate spectrophotometer (EL × 800UV, BioTek). As a result of ELISA analysis, optical density (OD)/concentration curves were created for each antibody using the standards. Using the OD values of the patient samples, the corresponding concentration values were calculated from the standard curve.

Statistical analysis

Statistical tests were selected according to the number of patients. All statistical analyses were performed using the SPSS for Windows 11.5 (SPSS Inc., Chicago, IL, USA). Comparisons of variables between groups were performed using Kruskal–Wallis test. A two-sided P < 0.05 was considered statistically significant.


  Results Top


General characteristics and symptoms of patients with coronavirus disease-2019

The general characteristics of the patients depending on the clinical conditions according to the different groups are given in [Table 2].
Table 2: General characteristics and clinical course of COVID-19-positive patients

Click here to view


The results of interleukin-17A serum concentrations

The IL-17A concentration level ranged between 2.45 pg/mL and 31.44 pg/mL and the highest IL-17A concentration was found at 31.44 pg/mL in a patient of Group 4. The mean values of IL-17A concentrations in the plasma samples of COVID-19-positive patients were found to be 3.945 pg/mL, 4.843 pg/mL, 5.015 pg/mL, and 7.412 pg/mL, respectively (P > 0.05).

The results of transforming growth factor-β 1 serum concentrations

No presence of TGF-β1 protein was found in the plasma samples of COVID-19 patients in Group 1 and Group 2. In Group 3 and 4 patients, some of the plasma samples were also negative. The TGF-β1 concentration level ranged between 72.46 pg/mL (the patient of Group 3) and 837.08 pg/mL and the highest TGF-β1 concentration was found in a patient of Group 4 (P > 0.05). The mean values of TGF-β1 concentrations were calculated as 126.211 pg/mL in Group 3 and 201.266 pg/mL in Group 4.

The results of interleukin-6 serum concentrations

No presence of IL-6 protein was found in the plasma samples of COVID-19 patients in Group 1, Group 2, and Group 3 [Figure 1]. In Group 4 patients, some of the plasma samples were also negative. The IL-6 concentration level ranged between 9.52 pg/mL and 526 pg/mL and the mean values of IL-6 concentration were determined as 77.141 pg/mL in Group 4 (P > 0.05).
Figure 1: The concentrations of IL-17A, TGF-β1 and IL-6 in COVID-19-positive patients depend on the groups (p>0.05). TGF-β1: Transforming growth factor-β 1, IL-17A: Interleukin-17A, IL-6: Interleukin-

Click here to view



  Discussion Top


The SARS-CoV-2 outbreak was declared a pandemic on March 11, 2020, by the World Health Organization (WHO).[10] Until August 3, 2020, there had been approximately 18,100,204 confirmed cases and 690,257 deaths reported to the WHO globally (https://covid19.who.int). The COVID-19 patients are characterized by high inflammatory parameters (cytokine storm) and reduced lymphocyte numbers.[10]

IL-17A is known as a mediator of respiratory infections by recruiting immune cells to produce other cytokines.[11] In viral infections, IL-17 is produced by Th17 cells in response due to the persistence of the virus and leads to the cytokine storm12. IL-6 is another important cytokine that is reported as promoting virus survival and inhibiting apoptosis during different viral infections. It is also known that IL-17 and IL-6 have synergistic behavior, which causes them to promote viral persistence and block apoptosis of infected cells together. Although the molecular mechanisms of IL-17 have not been confirmed, it is clearly observed that the higher levels of IL-17A are directly related to viral diseases and symptoms.[12] IL-17A also promotes hypercoagulation in deep-vein thrombosis pathogenesis.[13]

In this study, it was seen that the clinical course of the patients and elevated levels of IL-17A concentration were correlated. The increase of IL-17A enables the cell to escape from apoptosis in viral infections. Therefore, an increase in IL-17A may both support the survival of the virus and cause the triggered secretion of molecules such as other cytokines, chemokines, and growth factors, and this may then cause the clinical conditions to worsen.

When drug usage was evaluated in two COVID-19 patients with above average IL-17A levels (58,42 pg/mL and 13.61 pg/mL), it was observed that paracetamol, pantoprazole, and enoxaparin use were present in both patients. Therefore, it is thought that there may be a parallel relationship between these drugs and IL-17A levels. It is necessary to conduct detailed studies on this.

TGF-β family proteins stimulate cell differentiation, proliferation, motility, metabolism, and immune responses. In the virus-infected cells or tissues, the balance of TGF-β1 was changed and the levels of TGF-β1 were upregulated. It was shown in a study that the upregulation of TGF-β1 mediates pulmonary fibrosis of SARS pathogenesis.[14] TGF-β1 can be involved in the activation of Th17 cells while supporting the formation of IL-6 secretion from Treg cells. The absence of TGF-β1 secretion in Group 1 and Group 2, but the increase observed in some patients in Group 3 and Group 4, may lead to the aggravation of the clinical feature by controlling other factors involved in the continuing viral persistence and survival of the virus-infected cell. It is demonstrated that transmissible gastroenteritis virus (TGEV) significantly increased mRNA levels of specific cytokines IL-1 β, IL-6, TNF-α, IL-10, and TGF-β.[15] Furthermore, TGEV, which is a porcine virus, has been known to induce the production of cytokines and TGEV has similar symptoms to COVID-19. Qian et al. showed that TGEV infection stimulates TGF-β secretion.[16]

Our results demonstrated that TGF-β1 concentration was seen in some patients of Group 3 and Group 4. No presence of TGF-β1 protein was found in plasma samples of COVID-19 patients in Group 1 and Group 2.

The upregulated expression of TGF-β plays a key role in diseases such as respiratory diseases including asthma, lung cancer, and idiopathic pulmonary fibrosis. It mostly behaves as a suppressor of the immune system and increases the risks. TGF-β is secreted from many cell types including epithelial cells, eosinophils, macrophages, and fibroblasts. Furthermore, the aberrant levels of TGF-β are known to have the potential to suppress T-cells and induce apoptosis of B-cells.[17]

IL-6 is an important cytokine whose production is related to various inflammatory diseases. Coronavirus infectious bronchitis virus (IBV) is an enveloped RNA virus-like SARS-CoV-2 and causes ARD in chickens. In an in vitro study, it was shown that IL-6 as a pro-inflammatory cytokine was greatly elevated depending on the time in IBV-infected Vero and H1299 cell lines. The activation of IL-6 modulates the replication and pathogenesis of the virus in individual infected cells and changes the balance between apoptosis and cell survival.[18] Rossignol suggests the use of nitazoxanide, which is an antiviral drug that suppresses IL-6 production in mice, for the treatment of Middle East Respiratory Syndrome coronavirus and other coronaviruses and points to the suppression of overproduction of IL-6 as a factor that could the improve the outcomes of the disease.[19] Huang et al. reported that IL-2, IL-7, IL-10, granulocyte colony-stimulating factor, IP10, MCP1, MIP1A, and TNF-α were higher in severe COVID-19 patients.[20] It was also reported that IL-6 levels were higher in nonsurvival COVID-19 patients than in survivals.[21],[22]

In our study, elevated IL-6 secretion was only detected in some of the Group 4 patients who showed worse clinical conditions. In one patient of Group 4 with ex, IL-17A was well above the average (31,44 pg/mL) with the highest IL-6 concentration (526 pg/mL). This supports the survival of the virus-infected cell after the suppression of neutrophil function with the increase of IL-6.

The detection of higher levels of these cytokines in strong symptomatic SARS-CoV-2-positive cases showed that the accumulation of cytokine storm aggravates the clinical picture. The IL-17A, TGF-β1, and IL-6 targeted therapies can determine the control of SARS-CoV-2 infection. The inhibitors of cytokines or their receptors should be involved in the clinical trials during therapy.

SARS-CoV-2 is a single-chained enveloped RNA virus which causes different symptoms ranging from fever or headache to ARDS.[2] Cytokines are released from the virus-infected cells and in different patients, the clinical picture of this disease becomes different.[4] These various outcomes are directly related to cellular response. Different symptoms in COVID-19 patients may be observed after Th17 cell trigger and IL17A secretion, which is expressed from the lung, gut, or skin.[13] IL-17A may play a role as an upstream modulator of the inflammatory pathway in SARS-CoV-2-positive patients. Therefore, IL-17A inhibitor treatment may be chosen to decrease the virus infection and also secondary infections which cause tissue damage. In addition, after IL-17A secretion, the levels of both TGF-β1 and IL-6 increase and they support coronavirus survival in cells. It can suggest that all patients diagnosed as being SARS-CoV-2 positive should be screened for IL-17A, TGF-β1, and IL-6 levels and controlled during treatment to prevent acute lung injury or other organ problems. Uncontrolled immune dysregulation may cause after IL-6 secretion in COVID-19 disease, we suggest the targeted therapy for IL-6 secretion should be also chosen to worsen viral-infected patients. Determining the plasma cytokine levels of SARS-CoV-2-positive patients could be the answer to the symptomatic variety and could lead to the selection of an effective protocol for personal treatment.

SARS-CoV-2 has caused a large number of deaths with tens of thousands of confirmed cases worldwide, posing a serious threat to public health. In addition, to control the pandemic, both spread of viruses and the treatment of the patients will be important. Therefore, which cytokines secreted after SARS-CoV-2 infections and the response of the patients to them will be critical during therapy and reducing the death rate of COVID-19.

Financial support and sponsorship

The research was supported by the Near East University Scientific Research Projects Coordination Unit (project no: SAG2020-01-011).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Jean SS, Lee PI, Hsueh PR. Treatment options for COVID-19: The reality and challenges. J Microbiol Immunol Infect 2020;53:436-43.  Back to cited text no. 1
    
2.
Madabhavi I, Sarkar M, Kadakol N. COVID-19: A review. Monaldi Arch Chest Dis 2020;90:248-58.  Back to cited text no. 2
    
3.
Ahn DG, Shin HJ, Kim MH, Lee S, Kim HS, Myoung J, et al. Current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (COVID-19). J Microbiol Biotechnol 2020;30:313-24.  Back to cited text no. 3
    
4.
Acharya D, Wang P, Paul AM, Dai J, Gate D, Lowery JE, et al. Interleukin-17A promotes CD8+ T cell cytotoxicity to facilitate west nile virus clearance. J Virol 2017;91:e01529-16.  Back to cited text no. 4
    
5.
Hou W, Jin YH, Kang HS, Kim BS. Interleukin-6 (IL-6) and IL-17 synergistically promote viral persistence by inhibiting cellular apoptosis and cytotoxic T cell function. J Virol 2014;88:8479-89.  Back to cited text no. 5
    
6.
Sanjabi S, Oh SA, Li MO. Regulation of the immune response by TGF-β: From conception to autoimmunity and infection. Cold Spring Harb Perspect Biol 2017;9:a022236.  Back to cited text no. 6
    
7.
Mirzaei H, Faghihloo E. Viruses as key modulators of the TGF-β pathway; a double-edged sword involved in cancer. Rev Med Virol 2018;28:e1967.  Back to cited text no. 7
    
8.
Velazquez-Salinas L, Verdugo-Rodriguez A, Rodriguez LL, Borca MV. The role of interleukin 6 during viral infections. Front Microbiol 2019;10:1057.  Back to cited text no. 8
    
9.
Centers for Disease Control and Prevention. Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Patients Under Investigation (PUIs) for 2019 Novel Coronavirus (2019-nCoV). 31 January, 2020. Available from: https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-specimens.html. [Last accessed on 2020 Jul 03].  Back to cited text no. 9
    
10.
Zhang W, Zhao Y, Zhang F, Wang Q, Li T, Liu Z, et al. The use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (COVID-19): The perspectives of clinical immunologists from China. Clin Immunol 2020;214:108393.  Back to cited text no. 10
    
11.
Mahallawi WH, Khabour OF, Zhang Q, Makhdoum HM, Suliman BA. MERS-CoV infection in humans is associated with a pro-inflammatory Th1 and Th17 cytokine profile. Cytokine 2018;104:8-13.  Back to cited text no. 11
    
12.
Cafarotti S. Severe acute respiratory syndrome-coronavirus-2 infection and patients with lung cancer: The potential role of interleukin-17 target therapy. J Thorac Oncol 2020;15:e101-3.  Back to cited text no. 12
    
13.
Ding P, Zhang S, Yu M, Feng Y, Long Q, Yang H, et al. IL-17A promotes the formation of deep vein thrombosis in a mouse model. Int Immunopharmacol 2018;57:132-8.  Back to cited text no. 13
    
14.
Li SW, Wang CY, Jou YJ, Yang TC, Huang SH, Wan L, et al. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway. Sci Rep 2016;6:25754.  Back to cited text no. 14
    
15.
Xia L, Yang Y, Wang J, Jing Y, Yang Q. Impact of TGEV infection on the pig small intestine. Virol J 2018;15:102.  Back to cited text no. 15
    
16.
Qian S, Gao Z, Cao R, Yang K, Cui Y, Li S, et al. Transmissible gastroenteritis virus infection Up-regulates FcRn expression via nucleocapsid protein and secretion of TGF-β in porcine intestinal epithelial cells. Front Microbiol 2019;10:3085.  Back to cited text no. 16
    
17.
Lachapelle P, Li M, Douglass J, Stewart A. Safer approaches to therapeutic modulation of TGF-β signaling for respiratory disease. Pharmacol Ther 2018;187:98-113.  Back to cited text no. 17
    
18.
Liao Y, Wang X, Huang M, Tam JP, Liu DX. Regulation of the p38 mitogen-activated protein kinase and dual-specificity phosphatase 1 feedback loop modulates the induction of interleukin 6 and 8 in cells infected with coronavirus infectious bronchitis virus. Virology 2011;420:106-16.  Back to cited text no. 18
    
19.
Rossignol JF. Nitazoxanide, a new drug candidate for the treatment of Middle East respiratory syndrome coronavirus. J Infect Public Health 2016;9:227-30.  Back to cited text no. 19
    
20.
Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395:497-506.  Back to cited text no. 20
    
21.
Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet 2020;395:1054-62.  Back to cited text no. 21
    
22.
Vatansever HS, Becer E. Relationship between IL-6 and COVID-19: To be considered during treatment. Future Virol 2020;15:12,817-22. [doi: 10.2217/fvl-2020-0168].  Back to cited text no. 22
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed186    
    Printed0    
    Emailed0    
    PDF Downloaded36    
    Comments [Add]    

Recommend this journal