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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 31  |  Issue : 2  |  Page : 128-133

Estimation of the level of salivary proinflammatory cytokine in oral lichen planus – A case–control study in cuddalore-based population


1 Department of Oral Medicine and Radiology, RVS Dental College and Hospital, Coimbatore, Tamil Nadu, India
2 Department of Oral Medicine and Radiology, Rajah Muthiah Dental College and Hospital, Annamalai University, Chidambaram, Tamil Nadu, India
3 Department of Oral and Maxillofacial Surgery, Sri Ramakrishna Dental College and Hospital, Coimbatore, Tamil Nadu, India

Date of Submission23-Feb-2019
Date of Acceptance05-Jun-2019
Date of Web Publication24-Jun-2019

Correspondence Address:
Dr. J Nandhini
Department of Oral Medicine and Radiology, RVS Dental College and Hospital, Coimbatore - 641 402, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_43_19

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   Abstract 


Aim: To estimate and compare the proinflammatory cytokine [tumor necrosis factor (TNF)-α] level in the saliva of oral lichen planus (OLP) and controls. Materials and Methods: A total of 22 OLP cases who were clinically and histopathologically proven (12 male and 10 female patients, with a mean age of 42 years) and 22 controls matched for age, sex, and socioeconomic status were enrolled in the study. Salivary TNF-α level was analyzed by immunoassay kits. Statistical Package for Social Science version 21.0 software was used for data analysis. Results: The salivary TNF-α level was significantly higher (P < 0.001) in patients than in controls. Conclusion: The results suggest that TNF-α may be a good diagnostic biomarker for this potentially malignant disorder.

Keywords: Lichen planus, potentially malignant disorder, premalignant condition, proinflammatory cytokine, salivary diagnostics, TNF-α


How to cite this article:
Nandhini J, Ramasamy S, Kaul RN, Sakthivel S, Munshi M A, Sunil S. Estimation of the level of salivary proinflammatory cytokine in oral lichen planus – A case–control study in cuddalore-based population. J Indian Acad Oral Med Radiol 2019;31:128-33

How to cite this URL:
Nandhini J, Ramasamy S, Kaul RN, Sakthivel S, Munshi M A, Sunil S. Estimation of the level of salivary proinflammatory cytokine in oral lichen planus – A case–control study in cuddalore-based population. J Indian Acad Oral Med Radiol [serial online] 2019 [cited 2019 Nov 13];31:128-33. Available from: http://www.jiaomr.in/text.asp?2019/31/2/128/261090




   Introduction Top


Lichen planus (LP) was first described in 1869 as a complex immune-mediated mucocutaneous disease characterized by outbursts.[1] The disease has slight female predominance at a ratio of approximately 1.4:1, with a mean age in the fourth decade. The prevalence in India is around 2.6%. Oral lesions are chronic and rarely undergo spontaneous remission unlike the self-limiting nature of cutaneous lesions.[2] The World Health Organization (WHO) has grouped oral lichen planus (OLP) under the “potentially malignant disorders” (PMDs) of the oral mucosa[3] as the malignant transformation rate ranges between 0.4% and 5.3% with more than 5 years of follow-up.[4]

The pathogenesis of OLP has not been fully understood. Data collected during the past decade pointed to tumor necrosis factor (TNF)-α as a key cytokine in LP development and progression.[5] Simultaneously with the expression of other proinflammatory and anti-inflammatory cytokine, all the OLP lesions have been shown to contain cells with mRNA for TNF-α.[6] The 10- to 20-fold increase in salivary TNF-α levels in patients with LP[7] may be due to not only systemic production but also on a local basis.[8] It has been reported that the productivity of TNF-α is regulated by NF-κB, and the production of TNF-α can latter enhance the activation of NF-κB which leads to a coordinated increase in the expression of NF-κB-dependent cytokines, such as TNF-α, IL-6, IL-8, and chemokines, as well as the enzymes that mediate inflammatory and immune responses. This type of positive regulatory loop may amplify and perpetuate local inflammatory responses of OLP.[9] These activated T cells and cytokine production act locally and are not reflected in peripheral blood.[6]

High serum levels of TNF-α were detected in all patients with OLP in comparison to its hardly detectable levels in control subjects,[5] but salivary assay may be a more sensitive method to reflect the enhanced disease-related cytokine production when compared with serum and can be a good alternative to the latter.[10],[11]

However, due to inconsistent data and few available studies in the English literature that have evaluated TNF-α in saliva of patients with OLP, this study was undertaken to evaluate the salivary TNF-α levels in patients with OLP and to compare its level with the controls.


   Materials and Methods Top


Subjects' selection

This study was planned and conducted between June and September 2016 in the outpatient department of a dental college and hospital. After obtaining a formal ethical clearance to conduct the study from the ethical committee of the institution, a case–control study design was followed, and 22 patients with OLP and 22 controls were selected for participation in the study. On obtaining written informed consent, they were divided into two groups:

Group 1: 22 clinically and histopathologically proven cases with OLP

Group 2: 2 controls with age, sex, and socioeconomic status matched.

All the selected patients fulfilled the modified WHO diagnostic criteria of OLP and OLL[12] and age ranging between 15 and 75 years.

All other patients who underwent treatment for OLP within 6 weeks prior to the study, presence of other mucosal lesions, history of renal and liver diseases, psychiatric disorders, pregnancy, lactation and use of steroid hormone–based contraceptives, autoimmune, infectious diseases, history of trauma in the last 1 month, and persons who are not a native of Cuddalore district were excluded from the study.

A detailed case history was recorded for all patients in standard proforma. After establishing the clinical diagnosis of OLP [Figure 1], the patients were subjected to routine blood investigation to rule out any systemic ailments, followed by a histopathologic confirmation of diagnosis by performing punch biopsy of the lesion [Figure 2] and [Figure 3].
Figure 1: Clinical picture showing different types of LP. (a) Reticular. (b) Plaque-like. (c) Erythematous. (d) Ulcerative. (e) Actinic type. (f) Lichen planus pigmentosus

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Figure 2: Armamentarium for punch biopsy

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Figure 3: Histopathological picture of OLP showing parakeratinized stratified squamous epithelium with saw-toothed rete ridges and densely fibrous underlying stroma, with band of chronic inflammatory cell infiltration in the subepithelial region

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Saliva collection

The whole unstimulated saliva was collected before starting any dental treatment between 9:00 a.m. and 11:00 a.m. to avoid diurnal variation. The subjects were abstained from eating and drinking for at least 2 hrs prior to the sampling. All subjects were made to sit comfortably and 10 mL of saliva was collected according to spitting method described by Navazesh[13] in a sterile disposable plastic container and immediately centrifuged for 2 min at 10,000 rpm, and the clarified supernatant was separated using micropipette (1000 μL) in 2-mL aliquots (cryovials) and frozen at −80°C until assayed [Figure 4].
Figure 4: Armamentarium for saliva collection and supernatant separation

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Cytokine assay

All the reagents and samples were brought to room temperature and analyzed using enzyme-linked immune sorbent assay (ELISA) kit (Diaclone SAS, France) [Figure 5]. The standards were prepared according to the manufacturer's instructions. The test was performed in duplicates for reliability. The results were read using spectrophotometry at 450 nm and expressed as pg/mL.
Figure 5: TNF-α ELISA kit

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Statistical analysis

All the data were analyzed using Statistical Package for Social Science (SPSS) version 21.0 software. McNemar Chi-square test was used for pairwise comparison of the OLP group and control group variables. Paired Student's t-test was applied for comparison of the level of salivary TNF-α at baseline of the diseased individuals with the controls. P < 0.05 was considered to be statistically significant.


   Results Top


The study sample consists of 22 pairs of subjects involving 12 males and 10 females with age ranging between 19 and 72 years [Table 1]. Ulcerative OLP (36.4%) was the most common type of OLP [Graph 1] with the most affected site being the left buccal mucosa (77.27%) [Table 2].
Table 1: Demographic characteristics

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Table 2: Presence and absence of OLP based on anatomical site

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McNemar Chi-square test was used for pairwise comparison of the OLP group and control group variables with respect to the existing medical histories such as diabetes mellitus, hypertension, asthma, hypothyroid and peptic ulcer and adverse habits such as smoking, alcohol consumption and pan chewing which showed a nonsignificant statistical result (P > 0.05) indicating that there is no significant association existing between the cases, systemic diseases, and adverse habits [Table 3] and [Table 4].
Table 3: Distribution of the pairs of subjects and their medical history status

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Table 4: Distribution of the pairs of subjects and their adverse habits

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The salivary TNF-α level in patients with LP ranged from 18.9 to 82.3 pg/mL, with a mean ± SD of 54.18 ± 16.66 pg/mL, and in the control group it ranged from 9.7 to 39.6 pg/mL, with a mean ± SD of 22.08 ± 7.74 pg/mL. Paired Student's t-test showed that there was a significant difference (P < 0.001) in the level of salivary TNF-α at baseline of the diseased individuals with that of the controls [Table 5] and [Graph 2].
Table 5: Descriptive statistics of TNF-α groupwise

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   Discussion Top


TNF-αis a multifunctional cytokine that mediates inflammation, immune response, and apoptosis and also has a significant role in normal development and homeostasis of several organs.[14]

High concentration of TNF-α has a role in the progression of the pathological events in OLP. Serum or saliva level of TNF-αhas been used in some studies for monitoring the therapeutic response of OLP. A positive statistical correlation exists between TNF-αlevel in serum and saliva. Usage of saliva is increasingly preferred as saliva collection is noninvasive and more cost-effective.[11]

Smoking is a risk factor of oral precancerous and cancerous lesions.[15] However, there are no literature data indicating an elevated prevalence of OLP among smoking or alcohol consumption compared with the general population and controls;[16],[17] a similar finding was also observed in this study. Jureti et al.[18] reported that the salivary concentrations of TNF-α and IL-6 were not affected by smoking. Rhodus et al.[19] also found no significant difference in these cytokine concentrations between smokers and nonsmokers with oral cancer, OLP, and healthy controls.

There was no association between OLP and diabetes mellitus, hypertension, asthma, hypothyroid, and peptic ulcer observed in our study. Our results were supported by Jana et al.,[20] in which they observed mere coexistence of OLP with other systemic diseases such as diabetes mellitus, hypertension, hyperthyroidism, hypothyroidism, and peptic ulcer and play no direct role in the etiopathogenesis of OLP but present only as comorbidities. Bagewadi et al.[21] also agreed in their study that diabetes mellitus and hypertension do not play a direct role in the etiology of LP. The occurrence of LP-like lesions in the oral cavity could be due to the drugs given to the patients suffering from these systemic ailments.

This study demonstrated a statistically significant elevation in salivary levels of TNF-αin OLP samples when compared with controls. These data were consistent with their enhanced expression previously reported in studies from different media in patients with OLP.[22],[23] Our findings were also supported by other studies investigating the salivary levels of TNF-αin patients with OLP.[11],[19] Simark-Mattsson et al.[6] also have detected augmented mRNA levels of TNF-α in OLP tissues by PCR, supporting that TNF-α may play a crucial role in the pathogenesis of OLP and may lead to more specific and more effective therapies.

TNF-α is directed toward both tissue destruction and recovery. In malignant disease, high-dose local TNF-α selectively destroys tumor blood vessels, but when chronically produced, it may act as an endogenous tumor promoter, contributing to the tissue remodeling and stromal development necessary for tumor growth and spread.[24] The permanent or prolonged presence of high TNF-α amounts in saliva may contribute to malignant transformation of OLP lesions.[25] It is known that TNF-α has a role in carcinogenesis, as well as the ulcerative/erythematous form of OLP has a greater rate of malignant evolution in comparison to the reticular form of the disease. This point is supported by the studies by Kaur et al.,[17] Jureti et al.,[18] and Rhodus et al.,[19] who demonstrated an increase in salivary TNF-α level in an advanced stage of PMDs and in oral squamous cell carcinoma OSCC. Moreover, Pezelj-Ribaric et al. demonstrated significantly higher amounts of salivary TNF-αin patients with OLP compared with healthy controls which showed correlation with disease severity being significantly higher in the ulcerative/erythematous type than in reticular type of OLP which might be useful in monitoring disease activity.[26]

Considering that TNF-α has a role in carcinogenesis, particularly ulcerative and erythematous types having a greater rate of malignant evolution, finding early stage of the previously undetected disease may ultimately save lives. This stresses on the diagnostic and prognostic potential of salivary TNF-α in patients with OLP.

Although different clinical variants of OLP subjects were included in the study, a subgroup analysis on the correlation of OLP and TNF-α was not done.

We consider that future studies involving larger patient series in different populations can gain further insight about the role of TNF-α in the etiopathogenesis of OLP. Furthermore, pre- and posttreatment analysis of TNF-α can be done to evaluate the prognosis of the OLP.


   Conclusion Top


Increased salivary concentrations of TNF-α reflect its local production in PMD and in cancer tissues. Saliva presents a perfect medium to be explored for health and disease monitoring. Moreover, the use of easily accessible biomarkers may prove highly beneficial in large populations and in chemoprevention trials.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Jureti ć M, Cerovi ć R, Beluši ć-Gobi ć M, BrekaloPršo I, Kqiku L, Špalj S, et al. Salivary levels of TNF-α and IL-6 in patients with oral premalignant and malignant lesions. Folia Biologica 2013;59:99-102.  Back to cited text no. 18
    
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Rhodus NL, Cheng B, Myers S, Miller L, Ho V, Ondrey F. The feasibility of monitoring NF-αB associated cytokines: TNF-α, IL-1α, IL-6, and IL-8 in whole saliva for the malignant transformation of oral lichen planus. Mol Carcinog 2005;44:77-82.  Back to cited text no. 19
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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