Role of sToll-like receptors 2 and 4 in stage 2 periodontitis patients with and without type 2 diabetes: A Randomized clinical control trial

 

Amitha Ramesh¹, Sudhir Rama Varma^2,8, Srinivas Ramamurthy³, Maher al Shayeb^2,8,

Moyad Shahwan^4,8, Abed M. Atia Elkaseh^2,8, Al Moutassem Billah Khair^5,8, Adil Mageet^2,8,

Madeeha Arif⁶, Biju Thomas¹, Pavithra Jaganathan¹, Suchetha Shetty⁷, Sharmila K.P⁷

¹Department of Periodontics, A.B. Shetty Memorial Institute of Dental Sciences, Mangaluru, Karnataka, India.

²Department of Clinical sciences, College of Dentistry, Ajman University, Ajman, U.A.E.

³Department of Pharmaceutical Sciences, College of Pharmacy, University of Science and Technology of                           Fujairah, Fujairah, U.A.E.

⁴ Department of Clinical Sciences College of Pharmacy and Health Sciences, Ajman University, Ajman, U.A.E.

⁵ Department of Basic Sciences, College of Dentistry, Ajman University of, Ajman, U.A.E.

⁶ College of Dentistry, University of Science and technology of Fujairah, U.A.E.

⁷ Central Research Laboratory, Department of Biochemistry, K.S. Hegde Medical academy, Deralakattae,

Mangaluru, Karnataka, India.

⁸ Center for Medical and Bio-allied health sciences research, Ajman university, Ajman, U.A.E.

 

ABSTRACT:

Objective: The role of TLRs as principal signaling receptors in recognizing endotoxins on gram positive and negative bacteria is facilitated by TLRs and further enhances its role as a potential biomarker in assessing periodontal disease. The study aimed at evaluating the expression of sTLR2 and sTLR4 among healthy, periodontal and diabetic patients and further if there a comparable expression among the TLRs among genders. Material and Methods: Patients were selected according to pocket depth, Clinical attachment loss and radiographic bone loss. Unstimulated whole saliva was collected. sTLR2 and sTLR4 quantification was estimated by ELISA. Comparison of sTLRs between the study groups were performed using ANOVA followed by Tukey post Hoc test. Independent sample t test was used to compare between the genders. (p≤0.05) Result: Expression of sTLR2 was higher among periodontitis patients compared to diabetic patients, this was also evident with relation to genders. sTLR4 showed significant expression among the three groups and also among the genders. Conclusion: The expression of sTLR2 was higher among periodontitis patients compared to diabetic patients. More studies need to be carried out to evaluate TLRs values among genders.

 

 

 

 

 

INTRODUCTION:

Periodontal disease is a combination of multifactorial entities such as host immune response, bacterial virulence factors and causative factors that are directly and indirectly related to the causation of dysbiosis in the periodontal vasculature¹.  Impairment of homeostatic balance contributes to an accelerated inflammatory activity and responsible for dysbiosis of the biofilm complex^2,3. Invading pathogens are neutralized by Innate Immunity which contributes to the first line of defense in the host, this is achieved by selective recognition of specialized receptors termed pathogen associated molecular patterns (PAMPs), the detection of agents contributing to infection is achieved by Toll-like receptors (TLRs), whose role is critical against invading pathogens⁴.

 

The TLRs belong to a broad family of receptors categorized as pattern recognition receptors (PRRs). Cells of the innate immune system such as neutrophils, monocytes contain TLRs on their surface. Bacteria responsible for initiating inflammatory activity express PAMPs are localized by TLRs⁵. Upon recognition of a specific pathogen, specific member of the TLR family, upon activation initiates production of cytokine and adaptive immune response⁶. Currently, thirteen TLRs and their ligand have been identified^7,8,9. The TLRs are comprised of both membrane and soluble form, with the soluble form present in tears, saliva, serum and urine. The soluble TLRs (sTLRs) have inhibitory action with relation to membrane associated TLRs and are hypothesized to have negative regulatory function¹⁰. Among the TLRs family, the two TLRs that have been identified as principal signaling receptors are TLR2 and TLR4. Recognition of PAMPs, lipoproteins and peptidoglycans present on gram positive and negative bacteria is achieved by TLR2¹¹. Endotoxin such as lipopolysaccharide (LPS) and gingipains present on gram negative bacteria are recognized by TLR4¹².

 

It has been conceptualized that Porphyromonas gingivalis also referred to as the key stone pathogen^13,14, orchestrates polymicrobial synergy and dysbiosis in periodontal disease inflammation¹⁵. The transition from a symbiotic microflora to a predominantly gram negative dysbiotic flora is a synchronized step that features expression of select proteins and gene expression key to inflammatory and resorptive activity on the host cells, characteristic of any pathogenic resident microflora.   Microbial related stimulation results in gene expressions that have different TLR-mediated pathways^3-17. This further elucidates the fact that in periodontal tissue, the presence of TLRs, particularly TLR2 in connective tissue has been recorded. There is also presence of TLR4 and TLR9 in gingival epithelial cells and fibroblasts present in gingival and periodontal ligament cells^15,18,19. which determines the progression of periodontal disease in inflammatory conditions such as rheumatoid arthritis and diabetes.

 

Metabolic alterations that contribute to cardiovascular disease predominates with the presence of Diabetes Mellitus Type 2 which predominantly is responsible for insulin resistance, and inflammation²⁰. Most of the population who have Type 2 Diabetes Mellitus (T2DM), have increased prevalence of cardiovascular conditions²¹. The mortality rate increases with the uncontrolled progression of the condition²². One of the root causes for patients with T2DM is the presence of chronic hyperglycemia. This is mainly due to the inadequate release of Insulin coupled with insulin resistance²³. The increased modification of biochemical reactions together with protein and enzyme changes is associated with the presence of advanced glycation end products (AGEs) which contributes to the changes in tissue proteins further leading to diabetic complications²⁴. Periodontitis is considered one of the sixth complication of Diabetes which also includes microangiopathy, neuropathy, nephropathy, and delayed wound healing and macrovascular disease²⁵.

 

In Diabetic conditions more so with relation to T2DM, TLRs play a detrimental role in relation to insulin resistance, chronic hyperglycemia and contributes to atherosclerotic plaque buildup^20,26. Heat shock proteins 60,70, (HSP60,70), endotoxins, high mobility group B 1 protein (HMGB1P), AGEs and extracellular components are specific ligands present in TLR2 and TLR4 that bind to both gram positive and negative pathogens^27,28. The presence of both TLR2 and TLR4 in increased concentration in skeletal muscle tissue and adipose tissue of T2DM patients where it is characteristic for insulin resistance has been seen^27-30. Inflammatory pathways in systemic conditions plays a critical role in the comorbidities and etiological factors in T2D. Various animal studies have recently implicated the role of both TLR2 and TLR4 in cytokine production and consequently development of diabetes. The role of TLR4 in recent times has also been justified by its presence as an important bridge between innate immune system and free fatty acid Inflammation^31-33.

 

Though there are animal and human tissue data which suggest role of TLR2 and TLR4 in diabetic patients, especially T2DM³³. The presence of inflammation in diabetic patients due to alteration in TLR2 and TLR4 pathways need to be further understood. Periodontopathic bacteria is acted on by sTLR2 and sTLR4 initiated by oral epithelial cells. Apart from membrane bound receptors of TLR2 and TLR4, soluble forms of TLR2 and TLR4, (sTLR-2 and sTLR-4) play a detrimental role in binding to microbial receptors.  The purpose of our study is to determine whether there is a comparable difference between sTLR2 and sTLR4 values among male and female cohorts as no previous study has reported this variation and if any notable expression among sTLR2, 4 values among healthy, periodontitis and diabetic cohorts is seen. 

 

MATERIALS AND METHODS:

This interventional study was a randomized clinical trial. The duration of the study was 8 months, from May 2019 to December 2019. Informed consent was obtained from all the subjects, the study was approved by the Deanship of graduate studies and research ethical committee, Ajman University (Ref no- D-F-19-03-03), and it is also registered in Clinical Trials. gov PRS, ID no- NCT04201912. The research manuscript follows consolidated standards of reporting trials (CONSORT) guidelines as well as the Helsinki declaration for human research as revised in 2013.

 

Study Population:

The study population involved healthy, periodontal patients without diabetes and diabetic patients with periodontitis with an age group of 20-60, healthy (mean 35.467), periodontitis (36.893) and Diabetic with periodontitis (38.234). The assessment for grading and staging of periodontal diseases was done according to the criteria set by Tonetti et al³⁴.

 

Selection criteria for healthy subjects:

Inclusion Criteria-40 individual (n-20 male, n-20 female) with no clinical evidence of gingivitis or periodontitis were selected. Further patients with no family history of chronic systemic disease or diabetes and fasting blood glucose less than 100mg/dl were included in the study.

 

Exclusion criteria-Smokers, pregnant women, patients on antioxidants in the past three months or consumption of omega-3 fatty acid capsules were excluded from the study.

 

Patients with Periodontitis:

The study included 40 individuals (n-20 male, n-20 female) having clinical features of generalized moderate chronic periodontitis (CP) stage 2, (Clinical attachment loss CAL 3-4mm, probing depth of ≤ 5mm, it was validated by radiographic evidence of horizontal bone loss, and graded by calculating percentage of bone loss divided by age, 0.25-1 was categorized as grade B), the patients are systemically healthy and have no habits such as smoking, betel nut chewing. Patients who have undergone surgery in the past three months or under medication for any infection were also excluded from the study

 

Type 2 diabetes mellitus patients with Periodontitis:

Inclusion criteria for selection of T2DM patients with periodontitis clinically and radiographically were done using the same protocol as for patients with periodontitis without systemic condition, but inclusive factors were more elaborate considering medical factors. The study included 40 individuals (n-20 female, n-20 male) with controlled diabetes, HbA1C between 7-8%. Patients with thiazolidinedione, ACE inhibitors, angiotensin receptor blockers, insulin or statin were excluded from the study as they interfere with TLR expression.

 

Saliva collection:

Unstimulated whole saliva (UWS) was collected from the participating subjects according to procedure by prakasam et al³⁵. All the subjects were asked not eat or drink 1 hour prior to saliva collection. The UWS was collected by asking the subjects to tilt the head gently so as to initiate passive release of saliva. The position was maintained for a period of 15 minutes, whereby the saliva was collected in 15mL Eppendorf tube (Thomas Scientific, Swedesboro, NJ, USA). The samples were sent immediately to the laboratory in an ice box. The samples were centrifuged at 2000-3000rpm for 20 min to collect the supernatant.

 

Quantification of sTLR2 by ELISA:

ELISA for identification of sTLR2 is a double sandwich one-step assay. The assay range for identification employed was between 0.78ng/ml- 50ng/ml. A standard, test sample and HRP (Horse radish peroxide) labeled antibody were placed to enzyme wells which were pre-coated with TLR2 antibody, the incubation was carried out for a period of 1 hr and the uncombined conjugate was removed. 50ul of chromogen solution was added and incubated for 10min at 37 ⁰C, the color will change to blue, and further the reaction with acid (stop solution) causes the color to become yellow. The depth of the color and concentration was determined for a positive significance with relation to sTLR2.

 

The final measurement was carried out by setting the OD (Optical density) at 450nm wavelength and performed 15 min after adding the stop solution.

 

Quantification of sTLR4 by ELISA:

Identification for sTLR4 was also performed using a double sandwich enzyme linked immunosorbent one step assay (ELISA). The assay range was at 6.25ng/ml-200ng/ml. Similar to sTLR2, a standard, test and HRP labeled antibody wells were added to wells pre-coated with sTLR4 antibody, the incubation was carried out for a period of 1 hr at 37°C. 50ul of chromogen solution was added and incubated for a period of 10 min at 37°C. The color initially change to blue and then to yellow by adding the stop solution. Positive significance was determined for sTLR4 using depth and concentration.

 

The final measurement was carried out by setting the OD (Optical density) at 450nm wavelength and performed 15 min after adding the stop solution.

 

Statistical method:

Sample size was calculated keeping the confidence level at 95%, with a confidence interval at 1%. The population prevalence was assumed at 120 for a period of eight months. The sample size obtained was 119 which was rounded up significantly to 120. The data collected were entered into Microsoft excel spreadsheet and analyzed using IBM SPSS Statistics, Version 22(Armonk, NY: IBM Corp). Descriptive data were presented in the form of mean and standard deviation. Comparison of sTLR2 and sTLR4 between the study groups were performed using ANOVA followed by Tukey post Hoc Test. Independent sample t test was used to compare sTLR2 and sTLR4 between the genders. P value < 0.05 was considered as statistically significant.

 

RESULTS:

Comparison of sTLR2 and sTLR4 between the study groups:

sTLR2 levels were significantly higher in Diabetic (21.47±5.26) ng/mL and Non-Diabetic (23.32±3.73) ng/mL group as compared to Healthy subjects. (Table 1) There was no difference in TLR2 levels between the Diabetic and Non-Diabetic group.

 

Level of sTLR2 showed statistical significance between the three groups (p<0.001) with an interesting observation seen of the mean with relation to Periodontitis patients (23.32±3.73) ng/mL compared to diabetic patient (21.47±5.26) ng/mL. (Table 1, Figure 1) This could be attributed to TLR ligation, which is a trait normally seen in diabetic patients. There was significant expression of sTLR2 values between healthy and diabetic cohorts.

 

sTLR4 levels were significantly higher in Diabetic (96.75±10.76) ng/mL group followed by Non-Diabetic (73.62±15.54) ng/mL group and least in Healthy subjects (64.37±9.16) ng/mL. (Table 1, Figure 1)

 

sTLR4 showed a significant increase between the three groups with means significantly higher comparing diabetic (96.75±10.76) ng/mL and control cohorts (64.37±9.16) ng/mL. Means of cohorts in the periodontitis group (73.62±15.54) ng/mL also showed significant expression with diabetic patients. (Table 1)

 

Figure 1: Comparison of sTLR2 and sTLR4 among healthy, periodontitis and diabetic patients

 

Comparison of TLRs between study groups among gender:

sTLR2 among the male cohorts demonstrated significant expression among the three groups, with periodontitis patients exhibiting a mean (21.45±3.15) ng/mL higher than diabetic cohorts (19.07±4.19) ng/mL. (Table 2, Figure 2, 3) sTLR4 levels between the three groups in males showed significant expression with each other. There was significant variation between the TLRs among the male cohorts.

 

Among the three groups in the female cohorts, there was significant relation between the three groups with diabetic patients exhibiting a mean (23.87±5.20) ng/mL lower than periodontitis cohorts (25.20±3.35) ng/mL similar to males. (Table 2, Figure 2, 3)

 

Table 1: Comparison of TLR2 and TLR4 between the study groups

*p<0.005 Statistically Significant,                                               p>0.05 Non-Significant, NS

 

Table 2: Comparison of TLR2 and TLR4 between the study groups in each gender

*p<0.005 Statistically Significant,                                               p>0.05 Non-Significant, NS

 

 

There was statistically significant value among the three groups with relation to sTLR4. Comparing sTLR2 and sTLR4 between each other, there was significant relation.

 

Figure 2: Comparison of sTLR2 among Male and Female cohorts with healthy, periodontitis and diabetic patients

 

Evaluating TLRs between genders among study groups:

In the healthy cohorts in males, there was statistical significant relation to both sTLR2, with the healthy cohort exhibiting (11.33±3.30) ng/mL, periodontitis patients with (21.45±3.15) and diabetic patients having (19.07±4.19) and sTLR4, healthy cohort (59..88±5.80)ng/ml, periodontitis patients (70.97±18.55)ng/ml and diabetic patients with (93.66±13.34)ng/mL. (Table 2, Figure 3, 4) Among the females, sTLR2 levels among the healthy cohorts (14.02±3.02)ng/mL, periodontitis patients (25.20±3.35) and diabetic patients with (23.87±5.20)ng/mL, (Figure 3) with sTLR4 levels showing healthy with (68.86±9.81)ng/mL, periodontitis patients with (76.28±11.70)ng/mL and diabetic patients with (99.83±6.28)ng/mL. (Table 2, Figure 4, 5) sTLR2 levels in both diabetic and periodontitis cases, there was marked significance among both male and female cohorts. With relation to sTLR4, the values were non-significant among male and females in both periodontitis and diabetic patients.

 

Comparing sTLR2 and sTLR4 levels among healthy cohorts, there was significant expression with sTLR2 showing (p≤0.01) and sTLR4 showing (p≤0.001).(Table 3) Among the diabetic patients, sTLR2 showed a significant expression of (p≤0.001), but sTLR4 expressing a non-significant value (p≥0.29). In the periodontitis category, sTLR2 showed a significant expression of (p≤0.003) and sTLR4 showing a (p≥0.07) non-significant value. (Table 3)

 

Table 3: Comparison of TLR2 and TLR4 between the genders in each study group

*p<0.005 Statistically Significant, p>0.05 Non-Significant, NS

 

 

Figure 3: sTLR2 among Male and Female cohorts with healthy, periodontitis and diabetes patients

 

Figure 4: Comparison of sTLR4 among Male and Female cohorts with healthy, periodontitis and diabetic patients

 

Figure 5: Comparison of sTLR4 among Male and Female cohorts with healthy, periodontitis and diabetic patients

 

DISCUSSION:

Periodontal inflammation, proliferation of cell family and innate immunity initiation are detrimental roles played by TLRs^36,37,38. Bone metabolism and periodontal disease inflammation progression is believed to be orchestrated by sTLR2 and sTLR4^39,40. However, little is known about sTLR2 and TLR4 variation among gender. In the present study we evaluated whether there are significant differences among control, diabetic and periodontitis cohorts and among this population if there is any comparable distribution of sTLR2 and sTLR4 levels among genders.

 

Saliva was used to measure both sTLR2 and sTLR4 owing to the probability that both these TLRs function as potential deceptors for PAMPs. Elevated TLR2 levels has also been confirmed by immunofluorescent studies in periodontitis cases⁴¹. In our study there was significant expression of sTLR2 levels among healthy, periodontitis and diabetic cohorts. Contrary to some studies which was carried out among gingivitis patients, they reported a negative expression related to TLR2^41,42. In our study there was a positive expression in TLR 2 levels when comparing periodontitis cohorts (23.32±3.73) with diabetic patients (21.47±5.26). This could be attributed to the elevation of pathogen associated molecular pattern in the saliva of periodontitis patients (PAMPs), which is in agreement with a study by Lappin et al⁴³.

 

The probability of sCD14 in saliva influencing elevated production of sTLR2 in periodontitis patients is also a possibility and has been seen in various periodontal conditions^44,45. Saliva was used to measure both sTLR2 and sTLR4 owing to the probability that both these TLRs function as potential deceptors for PAMPs. Elevated TLR2 levels has also been confirmed by immunofluorescent studies in periodontitis cases⁴⁶. In our study T2DM (Type 2 diabetes mellitus) cohorts selected were controlled and the potential for an active periodontal disease progression was limited as validated by other studies^47,48. This could also be the reason for a reduced sTLR2 expression among diabetic patients in the presence of an inflammatory condition⁴⁹. The presence of elevated sTLR2 and sTLR4 levels in T2DM patients was elaborated by ex vivo studies done which involved gingival tissues from periodontitis patients^50-52. This further demonstrates the down signaling pathway of TLRs in an active inflammatory condition. Significant results for sTLR2 were demonstrated in our study while evaluating periodontitis cohorts with diabetic patients.

 

sTLR4 levels showed significant increase in expression from health to diabetes. The striking observation seen was the increase of sTLR4 in diabetic patients, though the HbA1C (glycated haemoglobin) levels were controlled for the period of the study. This could probably be due to an increased level of circulating endotoxins which contributes to a proinflammatory burden and spirals signaling pathways downwards⁵³. The presence of HSP (Heat Shock Protein) also contributes to the elevation in sTLR4 levels in T2DM by activating proinflammatory cytokines^54,55. Though some of the studies advocate the increased presence of HMGB1 (High Mobility Group Box 1) leading to increased presence of sTLR4, these studies have evaluated T1DM. In the present study, there could be a possibility of high titer of sTLR4 in T2DM diabetic patients as a result of an altered metabolism or due to a metabolic stressor contributing towards a pro inflammatory environment. It could also be due to nature of the salivary sample, quality, and disease status at the time of collection and comparable differences in the specificity and sensitivity of the antibodies added to the kit wells. Though majority of the patients having DM (Diabetes Mellitus) fall into the Type 2 category, it is still categorized as a proinflammatory state like T1DM. In a study done by Mythily et al where sTLR4 levels were elevated in gingivitis cases when compared to periodontitis cases, further corroborating the pro inflammatory status of T2DM⁴³. The hyper responsiveness of TLR4 in high glucose states in presence of microbial endotoxins like LPS (Lipopolysaccharides) also is a cause for elevated levels.

 

The literature regarding comparison of TLRs among genders with relation to periodontitis and diabetic cohorts is scarce. Maria et al recently reported no difference in TLRs profiles when comparing women with PCOS and control women. In the same study Fasting sTLR2 expression in neutrophils was higher in men than women⁵⁶. Contrary to this, in our study there was a positive correlation with sTLR2 expression, with values more in women when compared to males in both T2DM (p<0.001) and periodontitis cohorts (p< 0.003). With relation to sTLR4, there was a negative correlation with relation to both T2DM and periodontitis patients among female cohorts. From our study, we could presume that the elevated expression of sTLR2 in periodontitis was due to the presence of pro inflammatory activity working in a preliminary stage. We could also see that sTLR4 expression was influenced by the presence of glucose⁵⁶. sTLR2, sTLR4 levels showed significant difference when comparing T2DM and periodontitis cohorts with controls among both men and women. Reduced sTLR4 expression among both male and female represents a redemptive process, which is characteristic of LPS (Lipopolysaccharide) a characteristic endotoxin released by gram negative bacteria Aggregatibacter actinomycetemcomitans which is an auto shut mechanism seen in individuals already prone for further host bacterial inflammatory insults. This is characteristically seen in relation to sTLR2 and not TLR4^57-59. This could be one of the reason for a lack of congruence in our study comparing sTLRs results between healthy, T2DM and periodontitis cohorts. In addition sTLRs expression has been influenced by periodontal inflammation, dysbiosis due to host-bacterial interaction and sex hormones^60,61,62. Further it can be assumed that probably fasting among the cohorts could also have led to discrepancy in sTLR2 results in both men and women where in sTLR2 values greatly increased at the time of salivary sample delivery. In our study men presented with reduced sTLR4 expression when compared to females, probably owing to an inhibitory effect of testosterone on sTLR4 which was also in agreement with some studies^56,63. Contrary to a study by Jitprasertwong et al where men had higher expression of sTLR2 compared to females and  which indicated higher sTLR4 levels upon a pro inflammatory condition and reduced sTLR2 expression on estrogen influence⁶⁴. In our study the levels of sTLR2 in males were lower compared to females in both T2DM and periodontitis cohorts. Further studies evaluating the role of TLRs during menstrual cycle have also determined the presence of higher titers of sTLR2/sTLR4 ratio in the follicular phase compared to the luteal phase^65,66. This disparity in values of TLRs among women could be due to the presence of ectodomains created by splicing and proteolytic bifurcation⁶⁷. Further, the presence of lower sTLR2 has been seen in chronic diseases such as URTI( Upper respiratory tract infection) and more specifically colitis^68,69. A positive correlation of sTLR2 in periodontitis patients in both male and female cohorts and a direct correlation of sTLR4 in T2DM patients among both male and female cohorts suggest that both could function as a viable diagnostic marker.

 

The limitations in our study is given the complexity of our study design, we feel the sample size for each groups were relatively small to come to a conclusive hypothesis and probably enervated to detect changes within the groups. In our opinion, these limitations were compensated by the presence of a robust healthy cohorts, periodontitis patients and controlled T2DM patients. The results gathered from our study will open questions for the discrepancy in values found among male and female cohorts and the reasons which could have led to these changes.

 

CONCLUSION:

We observed an increase in sTLR2 levels in periodontitis patients when compared to diabetic cohorts among both male and female samples. There was positive correlation in both sTLR2 and sTLR4 levels when comparing female with male patients. Increase in sTLR4 levels were possibly due to relationship of TLR4 with endotoxin released by gram negative bacteria that are among the predominant microflora in both periodontal and diabetic patients.  These values could possibly suggest a transient discrepancy in actual readings or could be a normal immunologic response to prevent an increased inflammatory load, which was modulated by T2DM. Further investigations need to be carried out to rule out possible mechanisms in this disparity. Studies involving different parameters for female cohorts need to be designed to bring a possible explanation to these changes.

 

Data Availability:

The data used to support the findings of this study are included in the article.

 

CONFLICTS OF INTEREST:

The authors declare that they have no conflicts of interest.

 

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Received on 09.04.2020           Modified on 21.05.2020

Accepted on 30.06.2020         © RJPT All right reserved