Abstract
Background
In this study, cross‐reactive epitopes on β2 glycoprotein I and Saccharomyces cerevisiae have been described. The objective of our study was to determine the frequency of anti S. cerevisiae antibodies (ASCA) in patients with anti‐β2 glycoprotein I antibodies (aβ2GPI).
Methods
A retrospective study was conducted in 77 patients with aβ2GPI (aβ2GPI‐IgG or aβ2GPI‐IgA). Eighty blood donors were used as a control group. ASCA IgG and ASCA IgA were determined by Enzyme Linked Immunosorbent Assay (ELISA).
Results
Thirteen patients among 77 had ASCA. ASCA (IgA or IgG) was significantly more frequent in patients than in healthy subjects (16.9% vs. 3.7%, P = 0.01). The positivity of both ASCA IgG and ASCA IgA is higher in patients than in control group (6.5% vs. 0%, P = 0.02). The frequency of ASCA IgG was significantly higher in patients than in the control group (15.6% vs. 2.5%, P = 0.009). In females, the frequency of ASCA IgG was significantly higher in patients than in control group (17.5% vs. 3.7%, P = 0.03). The average titer of ASCA IgG was significantly higher in patients than in the control group (9.7 ± 23 U/ml vs. 2.2 ± 2.8 U/ml; P = 0.004). ASCA IgG was significantly more frequent than ASCA IgA in all patients (15.6% vs. 7.8%, P = 0.04).
Conclusion
The frequency of ASCA was significantly higher in patients with aβ2GPI than in the control group.
Keywords: anti‐β2 glycoprotein I antibodies, anti Saccharomyces cerevisiae antibodies, Tunisia
Introduction
Beta2 glycoprotein I (β2GPI), a cofactor of anticardiolipin antibodies, plays an important role in blood clotting and in immune response 1. Anti‐β2GPI antibodies (aβ2GPI) are potentially thrombogenic and responsible for several pathological complications 2, 3.
Anti Saccharomyces cerevisiae antibodies (ASCA), yeast commonly used in food industry, were considered as a serological marker for Crohn's disease 4. It has also been shown that ASCA had a high predictive value for inflammatory bowel disease 5. Additionally, elevated levels of ASCA had been found in patients with Behcet's disease 6, spondyloarthritis 7, coeliac disease 8, 9, intestinal tuberculosis 10, primary biliary cirrhosis 11, 12, autoimmune hepatitis 13, type 1 diabetes 14, and autoimmune thyroid disease 15, 16.
Recently we have demonstrated that ASCA were more frequent in patients with systemic lupus erythematosus (SLE) than in the control group 17. Furthermore, Krause et al. 18 described ASCA in primary antiphospholipid syndrome (APLS) and in patients with APLS associated with SLE. Moreover, these authors 18 have described cross‐reactive epitopes on β2GPI and S. cerevisiae. So, the aim of our study was to determine the frequency of ASCA in the Tunisian patients who have aβ2GPI and in whom SLE was excluded.
Patients and Methods
Patients
In our retrospective and multicenter study, we collected 77 sera samples, via the database of our immunology laboratory, from 77 patients (14 males, 63 females, median age 41 years, age range 24–83 years) with a positivity of aβ2GPI. Sera were collected between 2012 and 2013 from nine hospitals in the Center of Tunisia. Patients were admitted for suspicion of APLS. We cannot confirm that patients had APLS because we did not have the second sample for anti‐phospholipid antibodies (aPL) assay. SLE was excluded in all patients.
Sera of 80 blood donors (54 female and 26 male) were collected as normal controls. All sera were negative for aβ2GPI and were stored at −80°C until they were used. The study was approved by local Ethics Committee and all the patients gave their informed consent.
Methods
Anti S. cerevisiae antibodies
ASCA IgA and IgG were detected by a commercially available ELISA kit (Orgentec®, Mainz, Germany). The antigen contained highly purified mannan from S. cerevisiae. Results were expressed as arbitrary units with a cut‐off for positivity of 10 U/ml following the manufacturer's instructions.
aβ2GPI
IgG aβ2GPI and IgA aβ2GPI were determined by a commercial ELISA (Orgentec®) using a purified human β2‐glycoprotein I. Results were expressed as arbitrary units with a cut‐off for positivity of 8 U/ml following the manufacturer's instructions.
Statistical Analysis
The frequencies were compared using Chi‐square or Fisher's exact test. A P‐value less than 0.05 was considered significant.
Results
Frequency of ASCA in Patients and in the Control Group
Compared to the control group, patients had a significantly higher frequency of ASCA (IgG or IgA; 16.9% vs. 3.7 %, P = 0.01) and ASCA IgG (15.6% vs. 2.5%, P = 0.009). ASCA IgA was higher in patients than in the control group, but reaching a borderline significance (7.8% vs. 1.2%, P = 0.05). The positivity of both ASCA IgG and ASCA IgA is higher in patients than in the control group (6.5% vs. 0%, P = 0.02; Table 1).
Table 1.
Frequency of ASCA in Patients and in the Control Group
| Patients (n = 77) | Control group (n = 80) | P | |
|---|---|---|---|
| ASCA IgG or IgA | 16.9% (13/77) | 3.7 % (3/80) | 0.01 |
| ASCA IgG and IgA | 6.5% (5/77) | 0 % | 0.02 |
| ASCA IgG | 15.6%* (12/77) | 2.5% (2/80) | 0.009 |
| ASCA IgA | 7.8%* (6/77) | 1.2% (1/80) | 0.05 |
ASCA, anti Saccharomyces cerevisiae antibodies.
*P = 0.04.
Frequency of ASCA According to Gender
Thirteen patients among 77 had ASCA. Among these 13 patients, 12 were females. ASCA (IgG or IgA) was more frequent in females (19%) than in males (7.1%), but the difference was not statistically different. In females, the frequency of ASCA IgG was significantly higher in patients than the control group (17.5% vs. 3.7%, P = 0.03). The positivity of both ASCA IgG and ASCA IgA is higher in female patients than in the control group (7.9% vs. 0%, P = 0.04). In females, ASCA (IgG or IgA) were more frequent in patients than in the control group, but reaching a borderline significance (19.0% vs. 5.5%, P = 0.05; Table 2).
Table 2.
Frequency of ASCA According to Gender
| Female patients (n = 63) | Female subjects of control group (n = 54) | P | Male patients (n = 14) | Male subjects of control group (n = 26) | P | |
|---|---|---|---|---|---|---|
| ASCA IgG or IgA | 19% (12 /63) | 5.5% (3/54) | 0.05 | 7.1% (1/14) | 0% | NS |
| ASCA IgG and IgA | 7.9% (5/63) | 0% | 0.04 | 0% | 0% | NS |
| ASCA IgG | 17.5% (11/63) | 3.7% (2/54) | 0.03 | 7.1% (1/14) | 3.8% (1/26) | NS |
| ASCA IgA | 9.5% (6/63) | 1.8% (1/54) | NS | 0% | 3.8% (1/26) | NS |
ASCA, anti Saccharomyces cerevisiae antibodies; NS, not significant.
Comparison Between ASCA IgG and ASCA IgA
ASCA IgG was significantly more frequent than ASCA IgA in all patients (15.6% vs. 7.8%, P = 0.04; Table 1). ASCA IgG levels were significantly higher in patients than in the control group (9.7 ± 23 vs. 2.2 ± 2.8; P = 0.004), whereas ASCA IgA levels were almost equal in patients and the control group (Table 3).
Table 3.
ASCA IgG and IgA Levels in Patients and Control Group
| Patients (n = 77) | Control group (n = 80) | P | |
|---|---|---|---|
| ASCA IgG (U/ml) | 9.7 ±23 | 2.2±2.8 | 0.004 |
| ASCA IgA (U/ml) | 5.6 ±15.8 | 3.9 ±0.3 | NS |
ASCA, anti Saccharomyces cerevisiae antibodies; NS, not significant.
Frequency of ASCA and aβ2GPI Isotypes in 77 Patients
In patients, ASCA IgG was significantly more frequent than ASCA IgA (15.6% vs. 7.8%, P = 0.04). In contrast, aβ2GPI IgA was significantly more frequent than aβ2GPI IgG (83.1% vs. 22%, P ≤10−6; Table 4).
Table 4.
Frequency of ASCA and aβ2GPI Isotype in Patients
| aβ2GPI | ASCA | P | |
|---|---|---|---|
| IgG or IgA | 100% | 16.9% (13/77) | <10−6 |
| IgG and IgA | 6.5% (5/77) | 6.5% (5/77) | NS |
| IgG | 22% (17/77)* | 15.6% (12/77)** | NS |
| IgA | 83.1% (64/77)* | 7.8% (6/77)** | <10−6 |
*P <10−6.
**P = 0.04.
ASCA, anti Saccharomyces cerevisiae antibodies; aβ2GPI, anti‐beta 2 glycoprotein I antibodies; NS, not significant.
Characteristics of 13 Patients With ASCA
Among 77 patients with aβ2GPI (IgG or IgA), 13 have ASCA (IgG or IgA). Characteristics of these 13 patients are shown in Table 5.
Table 5.
Characteristics of 13 Patients With ASCA
| ASCA levels (U/ml) | aβ2GPI levels (U/ml) | ||||||
|---|---|---|---|---|---|---|---|
| N | Sex | Age (years) | IgG | IgA | IgG | IgA | Clinical manifestations |
| 1 | F | 30 | 100 | 35 | 14 | 68 | Venous thrombosis |
| 2 | F | 34 | 13 | 12 | 0 | 56 | Recurrent spontaneous abortions |
| 3 | F | 37 | 96 | 0 | 0 | 34 | Recurrent spontaneous abortions |
| 4 | F | 34 | 17 | 15 | 0 | 12 | Recurrent spontaneous abortions |
| 5 | M | 50 | 15 | 0 | 0 | 19 | Hypertension, stroke several times, and myocardial infarction |
| 6 | F | 37 | 22 | 6.3 | 0 | 18 | Recurrent spontaneous abortions |
| 7 | F | 58 | 20 | 8 | 0 | 13 | Purpura |
| 8 | F | 32 | 15 | 2.9 | 0 | 17 | Recurrent spontaneous abortions |
| 9 | F | 37 | 124 | 15 | 11 | 46 | Venous thrombosis, known with coeliac disease |
| 10 | F | 37 | 14 | 5 | 0 | 11.5 | Venous thrombosis |
| 11 | F | 42 | 32 | 134 | 0 | 70 | Venous thrombosis |
| 12 | F | 44 | 48 | 3.5 | 11 | 0 | Quadripyamidal syndrome, sphincter disorders |
| 13 | F | 29 | 4.1 | 28 | 0 | 27 | Recurrent spontaneous abortions |
ASCA, anti Saccharomyces cerevisiae antibodies; aβ2GPI, anti‐beta 2 glycoprotein I antibodies.
Discussion
The APLS is characterized by the presence of aPL that binds target molecules mainly via β2GPI 19, 20. The infectious origin of APLS has proved to be one of the explanations for generation of aβ2GPI 21. Indeed, among the environmental factors that trigger APLS, it is the bacteria, viruses, and yeast that were most described 21.
We have previously determined the frequency of ASCA in SLE 17. Krause et al. 18 determined the frequency of ASCA in patients with a primary APLS and in patients with APLS associated with SLE. In the present study, we determined the frequency of ASCA in patients with aβ2GPI and without SLE. In this study, ASCA (IgA or IgG) were significantly more frequent in patients than in healthy subjects (16.9% vs. 3.7%, P = 0.01). This frequency was similar to that found in the study by Krause et al. (20%) 18. Krause et al. explained this significant ASCA positivity in patients with APLS by cross‐reactivity between the two epitopes of antigens, namely phosphopeptidomannan of the yeast S. cerevisiae and the β2GPI. Recently, Rinaldi et al. 22 confirmed the result of Krause et al. 18 by consulting the protein database of the National Center for Biotechnology Information (NCBI) and they found a structural similarity of 39% between phosphopeptidomannan of the yeast S. cerevisiae and β2GPI.
In the study by Krause et al. 18, the frequency of ASCA (20%) was lesser than that of aβ2GPI (68.4%). Even in our study, ASCA were less frequent than aβ2GPI (16.9% vs. 100%). Indeed, only a subpopulation of aβ2GPI is specific to the glycosylated site of the β2GPI molecule that cross‐reacts with phosphopeptidomannan of S. cerevisiae 18. Furthermore, in our previous study, we determined the frequency of ASCA in SLE patients, and compared this frequency with that of aβ2GPI. The results showed a significantly lower frequency of ASCA (IgG or IgA) than that of aβ2GPI (IgG or IgA; 31.9% vs. 54.3%) 17.
The presence of cross‐reactive epitopes on β2GPI and S. cerevisiae, demonstrated in the study by Krause et al., suggests a pathogenic role of ASCA in Crohn's disease associated thrombosis 18. On the other hand, a significantly higher frequency of aβ2GPI has been reported in Crohn's disease patients compared with healthy subjects 23. However, it was not clear whether the patients with positive ASCA had or not an increased risk of thrombosis.
One patient, among 13 having ASCA, presented a myocardial infarction. It has been similarly reported between the sequence of phosphopeptidomannan and ICAM‐1. ICAM‐1 and P‐selectin are crucial molecules for transendothelial migration of leukocytes, playing an important role in the process of atherogenesis, especially in patients with a chronic systemic inflammatory autoimmune diseases 22. In a case‐control study whose purpose was to investigate the possible role of ASCA in atherosclerosis, high levels of IgA and IgG ASCA have been found in patients with acute myocardial infarction, suggesting that ASCA could be a useful marker of unstable atherosclerotic plaque 24.
In the present study, among 13 patients with ASCA, one was diagnosed with coeliac disease and having made several spontaneous abortions. In coeliac disease, there is an increased intestinal permeability that could be implicated in the genesis of ASCA 25. In fact, we and others 8, 9 demonstrated a high frequency of ASCA in coeliac disease. Furthermore, in another study we demonstrated a high frequency of aβ2GPI IgA in coeliac disease 26.
In the present study, the frequency of ASCA (IgG or IgA) was higher in female patients than in male (19% vs. 7%). Similarly, in our previous study on SLE, the frequency of ASCA (IgG or IgA) was higher in female patients (33%) than in male (23.1%) 17. These results could be explained by the fact that autoimmune diseases are more common in women because estrogen is implicated in the genesis of autoantibodies related to these pathologies 27.
In the present study, we found that the frequency of ASCA IgG was significantly higher than that of ASCA IgA (15.6% vs. 7.8%; P = 0.04) according to our previous studies 9, 12, 14, 16, 17 (Table 6).
Table 6.
ASCA IgG and IgA in Different Autoimmune Diseases
| Autoimmune diseases | ASCA IgG (%) | ASCA IgA (%) | P |
|---|---|---|---|
| Coeliac disease (9) | 24.8 | 8.8 | <10−6 |
| Primary biliary cirrhosis (12) | 18.9 | 1.6 | NS |
| Type 1 diabetes (14) | 21 | 9.8 | <0.002 |
| Graves’ disease (16) | 11.8 | 0.8 | 0.001 |
| SLE (17) | 57.5 | 7.5 | 10−3 |
| Present study | 15.6 | 7.8 | 0.04 |
NS, not significant.
In the present study, aβ2GPI IgA were significantly more frequent than aβ2GPI IgG (83.1% vs. 22%, P <10−6). The same result of the predominance of the IgA isotype was found in our previous studies 17, 26, 28 (Table 7). Indeed, it has been reported that IgA is the predominant isotype of aPL in African Americans 29.
Table 7.
aβ2GPI IgG and IgA in Different Autoimmune Diseases
| aβ2GPI IgA (%) | aβ2GPI IgG (%) | P | |
|---|---|---|---|
| SLE (17) | 50.9 | 19.8 | <10−6 |
| Coeliac disease (26) | 14.3 | 1.6 | 0.008 |
| Primary biliary cirrhosis (28) | 62.5 | 12.5 | <10−6 |
| Present study | 83.1 | 22 | <10−6 |
In conclusion, we and other described a high frequency of ASCA in many autoimmune diseases and in patients with autoantibodies. It remains to ascertain if ASCA is the cause or the consequence of these autoimmune diseases.
Conflict of Interest
None of the authors have conflicts of interest to declare.
Acknowledgment
This study is supported by: Unité de recherche: Auto‐immunité et Allergie (03/UR/07‐02), Faculté de Pharmacie de Monastir, Tunisia.
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