|Year : 2018 | Volume
| Issue : 2 | Page : 117-133
Studying Tenascin-C in systemic lupus erythematosus as a new biomarker for disease activity
Farag Khalil1, Nabil M Rafat1, Mohammad S Nada1, Mohammad Magdy2, Hassan A Abdelaziz3
1 Department of Internal Medicine, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
2 Department of Internal Medicine, Ministry of Health, Embaba Hospital, Giza, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
|Date of Submission||25-Jun-2018|
|Date of Acceptance||08-Oct-2018|
|Date of Web Publication||27-Feb-2019|
Department of Internal Medicine, 7th floor, Faculty of Medicine, Al-Azhar University, Alhusein University Hospital, Cairo, 11311
Source of Support: None, Conflict of Interest: None
Background The precise etiology and pathogenesis of systemic lupus erythematosus (SLE) remain unclear. Unpredictable flares and remissions and diverse serological and clinical manifestations are observed among patients with SLE and the challenge for the evaluation of disease activity and administration of appropriate treatment. Assessment of Tenascin-C (TNC) may reflect disease activity and/or early tissue damage in SLE.
Aim The aim of this study was to examine whether TNC levels are useful as a predictive biomarker in SLE and to reflect their activity.
Patients and methods In all, 50 patients with SLE (25 patients with active SLE, 25 patients with inactive SLE), and 25 age-matched and sex-matched healthy controls were enrolled in the study. Patients undergo clinical and laboratory assessment. Serum TNC was assessed by enzyme-linked immunosorbent assay.
Our results The results have shown that patients with active SLE had a higher TNC level compared with inactive patients and healthy volunteers. Also the study showed a statistically significant positive correlation between TNC level and Systemic Lupus Erythematosus Disease Activity Index score. On the other hand, the TNC level correlated negatively with white blood cells, platelet counts, C3 and C4 levels, hemoglobin level, and disease status.
Conclusion The increased serum tenascin level was found in patients with SLE and was correlated with certain clinical and laboratory immunoinflammatory parameters. So the estimation of serum Tenascin levels seems beneficial in the assessment of disease activity and progress in SLE patients as well as in the assessment of the efficacy of various treatment regimens used.
Keywords: disease activity, systemic lupus erythematosus, Tenascin-C
|How to cite this article:|
Khalil F, Rafat NM, Nada MS, Magdy M, Abdelaziz HA. Studying Tenascin-C in systemic lupus erythematosus as a new biomarker for disease activity. Al-Azhar Assiut Med J 2018;16:117-33
|How to cite this URL:|
Khalil F, Rafat NM, Nada MS, Magdy M, Abdelaziz HA. Studying Tenascin-C in systemic lupus erythematosus as a new biomarker for disease activity. Al-Azhar Assiut Med J [serial online] 2018 [cited 2020 Jul 6];16:117-33. Available from: http://www.azmj.eg.net/text.asp?2018/16/2/117/253094
| Introductions|| |
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by skin, kidney, lung, brain, heart, and joint manifestation. Its precise etiopathogenesis remains unclear. Although clinical assessment is the cornerstone of management of patients with SLE, these evaluations are limited and require additional instruments to confirm the diagnosis and determine disease activity. Diverse serological and clinical manifestations as well as unpredictable flares and remissions are observed among patients with SLE, and they present a challenge for the evaluation of disease activity and administration of appropriate treatment .
The lack of useful biomarkers for SLE hampers the assessment of disease activity and impedes the evaluation of treatment response. In spite of the traditional serological biomarkers such as anti-double-stranded DNA antibodies (anti-dsDNA antibodies) complement levels have been proven to be neither reliable indicators of disease activity nor predictors of impending disease flares. For this reason, there is growing interest in the exploration of new biomarkers for use as surrogate markers of disease activity and/or to predict flares of the disease .
Tenascin-C (TNC) is a large extracellular matrix glycoprotein that belongs to the damage-associated molecular patterns family. Little TNC is found in most healthy adult tissues, as they are specifically induced and tightly controlled during acute inflammation and are persistently expressed during chronic inflammation (Midwood et al. ).
The plasma levels of TNC have been shown to be useful indicators for chronic hepatitis C, inflammatory bowel disease, and myocarditis as their induction is highly associated with a wide range of diseases related to inflammation, including pneumonitis, hepatitis, inflammatory bowel disease, myocarditis, atherosclerosis, obesity, rheumatoid arthritis, and the enthesitis-related arthritis category of juvenile idiopathic arthritis. An early inflammatory response is generally associated with enhanced TNC levels, both in the plasma and in tissue (Catalán et al. ).
The circulating TNC levels could reflect disease activity and/or early tissue damage in SLE. Using clinical and laboratory data from our prospective cohort of patients with SLE, we investigated the association of serum TNC levels with Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2 K) scores and conventional laboratory markers of disease activity, such as anti-dsDNA, C3, C4, and antinucleosome antibodies. Moreover, we tested the clinical utility of serum TNC levels for the identification of patients with active disease and the prediction of disease flare (Brissett et al. ).
| Patients and methods|| |
Type of the study
A cross-sectional observational study.
Site and time of the study
Internal Medicine Department, Faculty of Medicine, Al-Azhar University. during the period from December 2016 to December 2017.
The study was conducted on 50 patients, 25 patients with active SLE, 25 patients with inactive SLE, 25 healthy individuals of matched age and sex as a control. All patients were women, their ages ranges from 18 to 40 years and the disease duration ranges from 6 months to 5 years.
Diagnosis of SLE was based on the American Rheumatism Association criteria for the classification of SLE (revised in 1997).
The activity of the disease was measured by the SLEDAI.
Participants in the study have been classified into three groups:
- Group I: 25 patients with active SLE.
- Group II: 25 patients with inactive SLE.
- Group III: 25 healthy individuals of matched age and sex as a control.
- Before data collection, verbal consent was granted from the ethics committee of Al-Azhar Faculty of Medicine.
- Informed consent was obtained from every patient to participate in this study.
- Proper treatment for diseased cases was prescribed.
SLE patients were diagnosed according to the SLE International Collaborating Clinics/American College of Rheumatology (SLICC/ACR) 2012 Criteria .
| Methods|| |
All participants were subjected to the following:
- Detailed history taking.
- Full clinical examination.
- Routine laboratory investigations. Erythrocyte sedimentation rate (ESR), C-reactive protein, fasting and 2 h postprandial blood glucose, complete blood count, complete urine analysis, liver and kidney function test.
- Measurement of proteins in 24 h urine (g/24 h).
- Antinuclear antibodies (ANA) and anti-dsDNA. Done by immunofluorescence technique. Titer of 1/40 or more is considered positive (done for SLE patients only).
- Serum complement levels (C3, C4): done by nephelometry (normal level of C3 is 84–160 mg/dl and for C4 12–36 mg/dl): done for SLE patients only.
- Specific laboratory investigation:
- Serum TNC by enzyme-linked immunosorbent assay.
- All standard, controls, samples, and plates are brought to room temperature prior to use.
- The required number of anti-tenascin-coated strips are placed in the frame provided. Reseal the unused strips in the foil bag along with the desiccant and refrigerate.
- Add 25 µl each of Tenascin standards, controls, and patient samples to the appropriate wells of the microtiter strip plate. Prepare wells with 25 µl of ‘0’ ng/ml standard and 100 µl of Tenascin enzyme diluent.
- Add 100 µl of diluted enzyme conjugate to all wells.
- Cover the plate and incubate at room temperature for 2 h on a rotary horizontal shaker (200 rpm is recommended).
- Aspirate contents of all wells or decant and blot plate on an absorbent paper.
- Wash wells three times with 200–300 µl of diluted wash buffer and aspirate or decant to dryness.
- Add 100 µl of color substrate to all wells and incubate for 30 min on a rotary horizontal shaker.
- Add 100 µl of stopping solution to all wells.
- Read absorbance at 450 nm and calculate the results using the supplied formula.
Statistical presentation and analysis of the present study
The quantitative data was described a mean±SD or median with interquartile range according to the normality of sample distribution, while qualitative data were described as frequency or percentage. Tests of associations for quantitative data were either one-way analysis of variance, independent t-test, or nonparametric Kruskal–Wallis test according to the normality of sample distribution. We used a χ2-test to compare the difference in quantitative data. Recessive operative curve was obtained to assess the diagnostic performance of TNC. Statistical Package for Social Science (SPSS) version 17 was used (IBM Corporation, 1 New Orchard Road Armonk, New York 10504-1722 United States). A P value of less than 0.05 was considered statistically significant ([Table 1],[Table 2],[Table 3],[Table 4],[Table 5]).
|Table 1 The relationship between serum Tenascin-C level and study groups|
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|Table 2 Recessive operative curve analysis for serum Tenascin-C between systemic lupus erythematosus patients and healthy control|
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|Table 3 The demographic and laboratory characteristics among study groups|
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|Table 4 The difference in 24 h protein and serum urea between study groups|
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| Results|| |
Moreover, the hemoglobinlevel and platelet counts were significantly lower in SLE patients compared with control groups (P<0.001 and 0.02, respectively) ([Table 6] and [Table 7]).
|Table 7 Systemic Lupus Erythematosus Disease Activity Index and anti-DNS between study groups|
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Our univariate correlation analysis showed a statistically significant positive correlation between TNC level and SELDAI score (r=0.764; P<0.001), 24 h urinary protein (r=0.749; P<0.001), ESR (r=0.707; P<0.001), and anti-DNS (β=6.65; P<0.001) ([Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8],[Figure 9],[Figure 10],[Figure 11],[Figure 12],[Figure 13],[Figure 14],[Figure 15],[Figure 16],[Figure 17],[Figure 18],[Figure 19]). On the other hand, the TNC level correlated negatively with white blood cells, platelet counts, C3 and C4 levels, hemoglobin level, and disease status ([Table 8] and [Table 9]).
|Figure 2 Recessive operative curve analysis showed a statistically significant area under the curve for the Tenascin-C level in systemic lupus erythematosus patients compared with healthy volunteers (area under the curve=0.992; P<0.001), which reflect the potential role of Tenascin-C as a diagnostic biomarker for systemic lupus erythematosus activity.|
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|Figure 7 The erythrocyte sedimentation rate level among the study group.|
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|Figure 10 Systemic Lupus Erythematosus Disease Activity Index score in the study groups.|
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|Figure 14 The correlation between Tenascin-C level and erythrocyte sedimentation rate.|
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|Figure 15 The correlation between Tenascin-C level and hemoglobin level.|
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|Figure 17 The correlation between Tenascin-C level and white blood cells.|
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|Figure 19 The correlation between Tenascin-C level and Systemic Lupus Erythematosus Disease Activity Index.|
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|Table 9 Associations between serum Tenascin-C levels of different groups and clinical and laboratory parameters|
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| Discussion|| |
The present study included female participants with active SLE, inactive SLE, or healthy volunteers. Our primary analysis showed that patients with active SLE had a higher TNC level (0.283±0.06) compared with inactive patients (0.074±0.055) and healthy volunteers (0.073±0.040). The difference was statistically significant (P<0.001). In concordance with our findings, Závada et al.  conducted a prospective cohort study on 59 SLE patients, and 65 healthy controls to evaluate the predictive role of TNC in SLE; and the patients were followed for a mean of 11 months. At the end of the study, patients with active SLE showed a statistically significant higher level of TNC compared with healthy controls; moreover, higher baseline levels of serum TNC presented a significantly greater risk of flare. The authors concluded that TNC seems to indicate the activity of SLE and may predict the need to escalate immunosuppressive therapy .
Serum TNC has been recently emerged as a useful predictor of disease activity and/or early tissue damage in SLE. TNC is upregulated under pathological conditions accompanying tissue injury and inflammation in many different organs that may be involved in the SLE disease process, including the joints, skin, kidney, lungs, heart, and central nervous system ,,. Therefore, TNC seems to be an eligible candidate surrogate marker of ongoing tissue damage and may reflect disease activity or an impending flare. Interestingly, our findings showed that TNC can act as a diagnostic biomarker for SLE activity (area under the curve=0.992; P<0.001). Similarly, Závada et al.  reported that TNC serum levels discriminated between active and inactive disease with an area under the curve of 0.69 (95% confidence interval: 0.53–0.86, P=0.02), a sensitivity of 53%, and specificity of 92%. Moreover, TNC expression was previously shown to have some applications in disease diagnosis and outcome prognostication in immune mediated and other inflammatory diseases, which further supports our findings ,,.
Our univariate correlation analysis showed a statistically significant correlation positive correlation between TNC level and SELDAI score and anti-dsDNA. Similarly, TNC levels were significantly associated with positivity of anti-dsDNA, antinucleosome antibodies, SLEDAI-2 K, and c-SLEDAI-2 K scores in a previous study .
With regard to patients’ demographic characteristics, patients with the active disease had a statistically significant higher degree of inflammation markers (WBCs and ESR) and lower value of complement components (C3 and C4) compared with the control group. In concordance with these findings, Charlesworth et al.  compared the metabolism of the complement proteins C3 and C4 between patients with active and inactive SLE, and found a statistically significant lower level of C3 and C4 levels in the active SLE group compared with the inactive group and healthy volunteers.
Complement is an important effector pathway of innate immunity and its role in the pathogenesis of SLE has been studied extensively ; the presence of the autoantibody, C3 nephritic factor, which causes C3 consumption, was hypothesized as the major mechanism for depressed level of C3 seen in patients with active SLE . Moreover, Birmingham et al.  suggested that C4 activation is critical for initiating renal flare while C3 activation is involved in the actual tissue damage, and that these effects are influenced by genetic variability in complement activation and regulation. Previous reports showed that the combined low level of C3 and C4 level can act as a biomarker of disease flare; however, the results of the currently published evidence is conflicting ,.
The SLEDAI is a clinical index for the assessment of lupus disease activity in the preceding 10 days which consists of 24 weighted clinical and laboratory variables of nine organ systems. The current body of literature indicates that the index has been shown to be reliable, has construct validity, and is sensitive to change in disease activity ,. Our analysis showed a statistically significant higher SLEDAI score among active patients than inactive patients.
Anti-dsDNA antibodies were reported to be hall markers for SLE and are the antibodies that initiate lupus glomerulonephritis; it has been also shown that a high level of anti-dsDNA correlates with disease activity and predicts flares in SLE . However, the clinical significance of anti-dsDNA antibodies has been reviewed at the 50th anniversary for the description of anti-dsDNA antibodies in SLE and was found to be largely nonspecific .
In the present study, 76% of the active patients in our study had positive anti-dsDNA compared with 28% in the inactive group (P=0.002). In agreement with our findings, Suleiman et al.  performed a cross-sectional study on 90 patients with established SLE to measure the levels of anti-dsDNA and antinucleosome antibodies; they found 60.9% anti-dsDNA antibodies in the active SLE patients compared with only 16.3% inactive SLE patients; the difference was statistically significant.
Lupus nephritis leading to severe persistent proteinuria, chronic renal failure, and end-stage renal disease remains one of the most severe complications of SLE and is associated with significant morbidity and mortality . Moreover, previous studies have shown a significant correlation between serum levels of interleukin-1β, interleukin-6, tumor necrosis factor-α, hyaluronan, and lipocalin and disease activity in patients with SLE which further highlights the importance of these inflammatory markers in the pathogenesis of lupus nephritis .
Our results have shown that the 24 h urinary protein and serum urea levels were significantly higher in active SLE patients compared with inactive patients and healthy volunteers. Similarly, Dolff et al.  found a statistically significant higher 24 h urinary protein and serum urea levels among active SLE patients compared with inactive patients. Another report by Du et al.  showed similar results.
To the best of our knowledge, this is one of the few published cohort studies which assessed the expression and predictive value of serum TNC in SLE patients. However, we acknowledge that the present study has a number of limitations: the number of the included patients was relatively lower than previous studies which affects the generalizability of our findings. Serum TNC levels were not assessed as different time points, so we could not assess the change in its levels with disease exacerbation and remission.
In conclusion, upregulation of serum TNC is significantly associated with active SLE and can act as a significant biomarker for disease activity. However, further well-designed studies are needed to confirm these findings.
The authors thank members of the laboratory in Al-Hussein University Hospital for their support in the preparation of this manuscript.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]