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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 37  |  Issue : 2  |  Page : 67-72

Measurement of the serum B-cell-activating factor of the tumor necrosis factor family (BAFF) in patients with idiopathic thrombocytopenic purpura and its correlation with immunosuppressive therapy


1 Department of Internal Medicine, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission21-Nov-2011
Date of Acceptance16-Jan-2012
Date of Web Publication23-Jun-2014

Correspondence Address:
Mervat A. Al-Feky
Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000415226.14757.90

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  Abstract 

Background

The B-cell-activating factor of the tumor necrosis factor family (BAFF) is a homotrimeric type 2 transmembrane protein that also exists in a soluble form. It belongs to the family of tumor necrosis factor ligands and is expressed at the surfaces of myeloid cells and antigen-presenting cells and induces B-lymphocyte proliferation and immunoglobulin secretion.

Aim of the study

The aim of this study was to assess the serum BAFF level in patients with idiopathic thrombocytopenic purpura (ITP) and to determine the correlation of its level with response to immunosuppressive therapy (corticosteroids).

Participants and methods

The study included 60 participants: 40 patients with newly diagnosed ITP who were followed up for 3 months after immunosuppressive therapy (steroids), divided into responders and nonresponders, and 20 healthy control individuals.

Results

The serum BAFF level was lower in the controls (mean±SD 3.4±2.2 ng/ml) than the ITP patients (responders and nonresponders) before treatment (mean±SD 19.8±2.9 and 20.5±8.8 ng/ml, respectively) with a statistically highly significant difference (P<0.001), but no significant difference between the responders and the nonresponders. After treatment, the BAFF level was still high in the nonresponder group (mean±SD 18±7.6 ng/ml), with a statistically highly significant difference in comparison with the responders and the controls (mean±3.6±1.9 and 3.4±2.2 ng/ml, respectively) (P<0.001). It was found that BAFF decreased markedly among all the participants and in the responder group, with a statistically highly significant difference between them and the nonresponder group. The percentage of change was 84% among responders compared with 10% only among nonresponders. In addition, the BAFF level was inversely correlated with the platelet count.

Conclusion

The serum BAFF level increases in patients with ITP and reverts to normal after treatment in responders but remains high in nonresponders. We recommend further randomized-controlled clinical trials exploring the role of drugs acting on BAFF in the treatment of autoimmune disorders.

Keywords: BAFF, B-lymphocyte stimulator, c-jun NH2-terminal kinase, immunosuppressive therapy, idiopathic thrombocytopenic purpura, nuclear factor-κβ, THANK


How to cite this article:
Soliman AA, Ayoub MS, Al-Feky MA, Tawfick GM, Kamal GM, El Bary HA. Measurement of the serum B-cell-activating factor of the tumor necrosis factor family (BAFF) in patients with idiopathic thrombocytopenic purpura and its correlation with immunosuppressive therapy. Egypt J Haematol 2012;37:67-72

How to cite this URL:
Soliman AA, Ayoub MS, Al-Feky MA, Tawfick GM, Kamal GM, El Bary HA. Measurement of the serum B-cell-activating factor of the tumor necrosis factor family (BAFF) in patients with idiopathic thrombocytopenic purpura and its correlation with immunosuppressive therapy. Egypt J Haematol [serial online] 2012 [cited 2019 Nov 17];37:67-72. Available from: http://www.ehj.eg.net/text.asp?2012/37/2/67/128292


  Introduction Top


Idiopathic thrombocytopenic purpura (ITP) is an autoimmune-specific disorder in which the platelets are opsonized by autoantibodies directed against platelet glycoproteins, followed by their destruction by macrophage 1. Although dysfunctions in antigen-presenting cell (APC) are considered to play crucial roles in ITP 2, the reason for the production of autoantibodies remains unknown and the clinical course is highly variable. Some patients remain asymptomatic, whereas others develop life-threatening bleeding episodes. Moreover, therapy including prednisolone, intravenous immunoglobulin G, anti-D, other immunosuppressive therapy, and splenectomy is not always effective, and only one-third of adult patients achieve long-term remission 3.

The B-cell-activating factor of the tumor necrosis factor family (BAFF) is a homotrimeric type 2 transmembrane protein that also exists in a soluble form. It belongs to the family of tumor necrosis factor (TNF) ligands.

BAFF is expressed at the surfaces of myeloid cells and APCs and induces B-lymphocyte proliferation and immunoglobulin secretion. It binds with high affinity to three types of receptors: B-cell maturation antigen; a transmembrane activator and calcium modulator, cyclophilin ligand-interactor (TACI); and the BAFF receptor (BAFF-R or BR3). The homolog molecule APRIL binds to TACI and B-cell maturation antigen but not to BAFF-R. TACI is activated by multimerized or membrane-bound BAFF and multimerized APRIL, but not by homotrimers 4. BAFF plays an important role in humoral immunity and T-cell-independent type II responses require the interaction of BAFF 60-mer or membrane BAFF with TACI 5.

The activation of intracellular Toll-like receptors in B cells by immune complexes containing nucleic acids upregulates the expression of BAFF-Rs, particularly TACI, and increases B-cell receptor-mediated signaling. In contrast, activation of B cells through toll-like receptor 4 upregulates BAFF-R preferentially and renders the cells sensitive to Fas-mediated apoptosis 6. An excess of BAFF can drive the development of autoimmunity through activation of the innate immune system alone. So, BAFF may help perpetuate disease in a manner that is independent of cognate T-cell help. Some studies have shown that activation of human memory B cells can be driven by the combination of BAFF, cytosine–guanine dinucleotide ligation, and cytokines. Thus, generalized inflammation and high levels of BAFF may drive continued production of plasma cells, producing pathogenic autoantibodies 7,8.

BAFF is elevated in the serum of patients with rheumatoid arthritis 9 and Sjögren’s syndrome 10, and it is suggested to play a role in the pathogenesis of multiple sclerosis 11 and systemic lupus erythematosis (SLE) 12.


  Aim of the study Top


The aim of this study was to determine the B-cell-activating factor of the tumor necrosis factor family (BAFF) in the serum of patients with ITP and assess its correlation with response to immunosuppressive therapy (corticosteroids).


  Participants and methods Top


Participants

The study included 60 participants: 40 patients with ITP, selected from the Clinical Hematology and Oncology Unit (Ain Shams University Hospital) who were followed up for 3 months after immunosuppressive therapy (corticosteroids), and 20 healthy control individuals.

Exclusion criteria

Patients with systemic autoimmune disorders such as SLE, rheumatoid arthritis, and Sjögren’s syndrome were excluded from the study, as BAFF levels may be elevated in the serum of these patients, as well as patients with secondary causes of ITP such as HCV infection and some types of cancers such as non-Hodgkin lymphoma and chronic lymphocytic leukemia.

All the patients and control participants were subjected to the following:

  1. Full history and physical examination.
  2. Laboratory investigations including complete blood count using a cell counter (CELL-DYN 1800; Abott Diagnostics, Abott Park, Illinois, USA.) together with examination of a Leishman-stained peripheral blood film to ensure true thrombocytopenia, reticulocyte count (manual count using brilliant-cresyl blue staining), and full liver and kidney chemical panel using an automatic analyzer (Hitachi 917; Roche).
  3. Serum BAFF was assessed in all patients at the time of diagnosis and in the control group, and was repeated for patients 3 months after immunosuppressive therapy.


In addition, patients were subjected to the following: assessment of viral markers (hepatitis B surface antigen, hepatitis C antibody, HIV antibodies, Epstein–Barr virus antibodies) and collagen markers (antinuclear antibody, anti-double-stranded DNA), Coomb’s test (direct and indirect), pelviabdominal ultrasound, and bone marrow (BM) aspiration with a morphological assessment using Leishman-stained BM smears to confirm the diagnosis of ITP.

Sample collection and storage

A volume of 10 ml of venous blood was collected from the controls and the patients in EDTA, sodium citrate-containing tubes (for complete blood count and Erythrocyte sedimentation rate; ESR) and plain tubes for chemical tests and BAFF assessment. An extra 5 ml of venous blood for immunological markers was collected and BM aspiration, to verify the diagnosis of ITP, was carried out for all patients according to the established protocol in Ain Shams University Hospitals.

From the sample collected for BAFF measurement, serum was rapidly separated and collected in plastic tubes after clotting and rapid centrifugation of the samples. Samples were stored at −20°C until the test run.

Methods

BAFF measurement was performed using human BAFF, instant ELISA (BMS2007INST; Bender MedSystems; Campus Vienna Biocenter 2, A-1030 Vienna, Austria, Europe). The kit was stored at 20°C until the time of use.

A well-detailed method is included in the kit; in brief, 100 μl of distilled water was added to the sample wells and different volumes to all standards and blanks according to the label of the standard strips. Then, 50 μl of each sample was added, mixed, covered with a plate cover, and incubated at room temperature for 3 h with shaking. After incubation, the sample was washed six times, followed by complete removal of the washing buffer. Then 100 μl of substrate solution was added to all wells, including the blank, followed by incubation in the dark at room temperature for 10 min. After incubation and color development, 100 μl of stop solution was added quickly and finally read using an ELISA reader at 450 nm.

A standard curve was prepared from seven human BAFF standard dilutions and human BAFF sample concentrations were determined. Normally, the range of the BAFF level varies from not detectable to 0.8 ng/ml.

Statistical analysis

Analysis of data was carried out by an IBM computer using statistical program for social science version 12 (SPSS; SPSS Inc., Chicago, Illinois, USA).


  Results Top


This study included 40 patients with ITP (35 women and 5 men) and 20 controls (10 women and 10 men). Among the patients, 34 responded to treatment but six patients were nonresponders. The disease was more common among women with a highly significant correlation between female sex and the lack of response to treatment (6/6; 100% of nonresponders were women) [Table 1].
Table 1: Comparison of the studied groups in terms of sex

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Platelets showed a statistically significant difference between the patients and the controls before and after treatment, with significantly lower mean values among the cases [Figure 1].
Figure 1: Changes in platelets before and after treatment among cases.

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The serum BAFF level was significantly higher in patients before treatment in comparison with its level in the controls and patients after treatment, with the mean values being 19.9, 3.4, and 5.7 ng/ml, respectively [Table 2]. In addition, the BAFF level was significantly higher in nonresponders in comparison with responders after treatment, with the mean values being18 and 3.4 ng/ml, respectively [Table 2] and [Figure 2], and the percentage of change in serum BAFF was significantly highly different between the responders and the nonresponders [Table 3]. Serum BAFF was inversely correlated with the platelet count as shown in [Figure 3] and [Figure 4] and showed higher sensitivity rather than specificity in correlation with the prognosis [Table 4] and [Figure 5]. BAFF is considered better positive screening marker than negative and is considered a valid marker with a cut off value 15 ng/ml. When BAFF level is above 15 ng/ml, it can predict good response to treatment, while if less than 15 ng/ml it can predict less response ([Table 4]).
Table 2: Comparison of the cases and the controls in terms of BAFF before and after treatment

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Table 3: Changes in BAFF before and after among the studied groups

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Table 4: BAFF validity as a prognostic factor in idiopathic thrombocytopenic purpura (ITP) cases

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Figure 2: Comparison of the studied groups in BAFF before and after treatment.

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Figure 3: Correlation between BAFF before treatment versus platelets before treatment among (a) responders and (b) nonresponders.

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Figure 4: Correlation between BAFF versus platelets after treatment among (a) responders and (b) nonresponders.

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Figure 5: ROC curve showing the validity of BAFF as a prognostic factor in idiopathic thrombocytopenic purpura cases.

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  Discussion Top


The B-cell-activating factor of the tumor necrosis factor family (BAFF) [also known as B-lymphocyte stimulator, a TNF homolog that activates apoptosis (THANK), nuclear factor-κβ (NF-κβ), c-jun NH2-terminal kinase] is a homotrimeric type 2 transmembrane protein that also exists in a soluble form. It belongs to the family of TNF ligands 4.

Schneider 13 found that BAFF is expressed at the surfaces of myeloid cells and APCs and induces B-lymphocyte proliferation and immunoglobulin secretion. Macrophages and dendritic cell-derived BAFF are key molecules by which these APCs regulate human B-cell proliferative responses to T-cell-independent stimuli.

Thien and colleagues found that BAFF plays a crucial role in B-cell development, survival, and immunoglobulin production. Moreover, excess amounts of BAFF result in the rescue of self-reactive B cells from anergy, thus implicating a role in the development of autoimmunity 14.

In the present study, BAFF was measured in the serum of patients with ITP and was correlated with the immunosuppressive therapy (corticosteroids). It was measured in 40 ITP patients, 35 (85.3%) women and five (14.7) men, and is in accordance with the data obtained by Segal and Powe, who found that women have a higher incidence of ITP compared with men. Of the 454 patients in their study, 62% were women, with an overall prevalence rate ratio of 1.9 for women to men 15.

According to Rodeghiero and colleagues, a platelet count less than 100×109/l was established as the threshold for the diagnosis of thrombocytopenia in cases of primary ITP. In addition, ‘complete response’ is defined as any platelet count of at least 100×109/l, ‘response’ is defined as any platelet count between 30 and 100×109/l and at least doubling of the baseline count, and finally ‘no response’ is defined as any platelet count lower than 30×109/l or less than doubling of the baseline count. The definition of a response also requires a concurrent resolution of bleeding symptoms 16.

In this study, the mean platelet count at presentation in all cases was 15.5±7.8 compared with the control group, with 279±65, and the mean platelet count after treatment was 288±65 in the responder group compared with only 34±8 in the nonresponder group.

Serum BAFF levels in untreated patients with ITP in this research were significantly higher (19.9±4 ng/ml) than those in the healthy controls (mean±SD 3.4±2.2 ng/ml), with a statistically highly significant difference between them. In addition, the BAFF level was the lowest among the controls (3.4±2.2 ng/ml) compared with both the groups (responders and nonresponders) before treatment (19.8±2.9 and 20.5±8.8 ng/ml, respectively), with a statistically highly significant difference between them (P<0.001), but no significant difference between the responders and the nonresponders.

This is in accordance with the data obtained by Emmerich and colleagues, who found that serum BAFF levels in patients with untreated ITP were significantly higher (median: 1620 pg/ml; range: 767–5821) than those in healthy individuals (median: 977 pg/ml; range: 470–1382; P<0.001), and the BAFF levels in responders were observed to be similar to those of the healthy control individuals (median: 1022 pg/ml; range: 545–3614) 3.

Similar data was obtained by Zhu et al. 17, who found that the level of plasma BAFF in ITP patients with active disease was significantly higher (593.1±219.0 pg/ml) than that in patients in remission (432.5±121.4 pg/ml, P<0.05) and controls (454.4±132.5 pg/ml, P<0.05). No significant difference was found between patients in remission and healthy controls (P>0.05). Wang et al. 18 also found that the plasma BAFF level (599.70±199.40 pg/ml) was significantly increased (P<0.05) in ITP patients before treatment as compared with that in controls (454.5±132.5 pg/ml).

In the current study, BAFF was found to be markedly decreased in all the participants and the responder group after treatment whereas it was still high in the nonresponder group (18±7.6 ng/ml) in comparison with the responders and the total cases (3.6±1.9 and 5.7±6 ng/ml, respectively), with a statistically highly significant difference (P<0.001). The percentage of change in BAFF level before and after treatment was 84% among the responders compared with 10% among the nonresponders. This is also in agreement with the results of Wang et al. 18, who found a significant decrease in the level of plasma BAFF after treatment with dexamethasone (296.9±119.7 pg/ml, P<0.01).

In terms of the relation between the BAFF level and the platelet count, this study showed that there was a statistically significant inverse correlation between the BAFF level and the platelet count before and after treatment in both responders and nonresponders.

The mechanism underlying the relationship between excess BAFF and platelet counts remains unclear. Hase et al. 19 showed that BAFF enhances both the expression of CD19 and the ability of the B-cell receptor to enhance and phosphorylate CD19. Moreover, Thien et al. 14 showed that BAFF mediates the maturation of autoreactive B cells. Thus, excess BAFF may play a role in promoting the accumulation of self-reactive B-cell clones directed against platelets.

The nature of these immune abnormalities and the important role that BAFF plays in autoimmunity led Zhu et al. 17 to examine BAFF in patients with ITP. They measured the BAFF levels in 45 patients with chronic ITP and found that patients with active disease had higher levels of plasma BAFF and BAFF mRNA than patients in remission and controls. Using in-vitro assays, they found that the addition of recombinant human BAFF to the culture not only promoted the survival of CD19+ B cells and CD8+ T cells but increased the apoptosis of platelets and the secretion of IFN-γ. They then examined how an inhibitory BAFF-receptor-Fc fusion protein (BR3-Fc) affected the above responses and found that BR3-Fc successfully corrected the above effects of recombinant human BAFF. These findings suggest that not only does BAFF play a pathogenic role in ITP by promoting the survival of both B and T cells, but blockade of BAFF signaling by BR3-Fc might be a promising therapeutic approach for ITP.

In this study, BAFF is considered better positive screening marker than negative and is considered a valid marker with a cut off value 15 ng/ml. When BAFF level is above 15 ng/ml, it can predict good response to treatment, while if less than 15ng/ml it can predict less response.

The current treatment strategies in ITP include the administration of immunosuppressive substances such as glucocorticoids, methotrexate, and azathioprine. Aggarwal 20 showed that the BAFF promoter is controlled by NF-κβ, with NF-κβ activity suppressed by several immunomodulators including corticosteroids and methotrexate, and Emmerich et al. 3 showed that BAFF was suppressed to normal levels in patients receiving these substances. Therefore, it is possible that transcriptional downregulation of BAFF represents a novel mechanism by which these substances exert their therapeutic effects. Also, immunosuppressive substances may act indirectly on BAFF expression through downregulation of these factors. However, it cannot be excluded that certain drugs bind directly to the BAFF promoter, thereby suppressing its activity.

Belimumab is a fully human monoclonal antibody that specifically recognizes and inhibits the biological activity of BAFF and few phase III trials have shown that belimumab in combination with standard of care significantly reduced SLE disease activity and SLE flare rates in patients with active SLE 21. Further trials using such drugs should be carried out to prove their effectiveness and safety in cases of autoimmune disorders.


  Conclusion Top


The serum BAFF level increases in cases of ITP and reverts to normal after treatment in responders but remains high in nonresponders, with a cut-off positive value 15 ng/ml. We recommend further randomized-controlled clinical trials exploring the role of the mentioned agents in the treatment of autoimmune disorders.[21]

 
  References Top

1.George JN, Raskob GE, Vesely SK, Moore D Jr, Lyons RM, Cobos E, et al. Initial management of immune thrombocytopenic purpura in adults: a randomized controlled trial comparing intermittent anti-D with routine care. Am J Hematol. 2003;74:161–169  Back to cited text no. 1
    
2.Yang Q, Xu S, Li X, Wang B, Wang X, Ma D, et al. Pathway of Toll-like receptor 7/B cell activating factor/B cell activating factor receptor plays a role in immune thrombocytopenia in vivo. PLoS One. 2011;6 : e22708  Back to cited text no. 2
    
3.Emmerich F, Bal G, Barakat A, Milz J, Mühle C, Martinez Gamboa L, et al. High-level serum B-cell activating factor and promoter polymorphisms in patients with idiopathic thrombocytopenic purpura. Br J Haematol. 2007;136:309–314  Back to cited text no. 3
    
4.Bossen C, Cachero TG, Tardivel A, Ingold K, Willen L, Dobles M, et al. TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts. Blood. 2008;111:1004–1012  Back to cited text no. 4
    
5.Von Bülow GU, van Deursen JM, Bram RJ. Regulation of the T-independent humoral response by TACI. Immunity. 2001;14:573–582  Back to cited text no. 5
    
6.Acosta Rodríguez EV, Craxton A, Hendricks DW, Merino MC, Montes CL, Clark EA, et al. BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas-mediated cell death. Eur J Immunol. 2007;37:990–1000  Back to cited text no. 6
    
7.Ettinger R, Sims GP, Robbins R, Withers D, Fischer RT, Grammer AC, et al. IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from a unique population of human splenic memory B cells. J Immunol. 2007;178:2872–2882  Back to cited text no. 7
    
8.Doreau A, Belot A, Bastid J, Riche B, Trescol Biemont MC, Ranchin B, et al. Interleukin 17 acts in synergy with B cell-activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus. Nat Immunol. 2009;10:778–785  Back to cited text no. 8
    
9.Bosello S, Youinou P, Daridon C, Tolusso B, Bendaoud B, Pietrapertosa D, et al. Concentrations of BAFF correlate with autoantibody levels, clinical disease activity and response to treatment in early rheumatoid arthritis. J Rheumatol. 2008;35:1256–1264  Back to cited text no. 9
    
10.Daridon C, Devauchelle V, Hutin P, Le Berre R, Martins Carvalho C, Bendaoud B, et al. Aberrant expression of BAFF by B lymphocytes infiltrating the salivary glands of patients with primary Sjögren’s syndrome. Arthritis Rheum. 2007;56:1134–1144  Back to cited text no. 10
    
11.Dalakas MC. B cells as therapeutic targets in autoimmune neurological disorders. Nat Clin Pract Neurol. 2008;4:557–567  Back to cited text no. 11
    
12.Groom JR, Fletcher CA, Walters SN, Grey ST, Watt SV, Sweet MJ, et al. BAFF and MyD88 signals promote a lupus-like disease independent of T-cells. J Exp Med. 2007;204:1959–1971  Back to cited text no. 12
    
13.Schneider P. The role of APRIL and BAFF in lymphocyte activation. Curr Opin Immunol. 2005;17:282–289  Back to cited text no. 13
    
14.Thien M, Phan TG, Gardam S, Amesbury M, Basten A, MacKay F, et al. Excess BAFF rescues self-reactive B cells from peripheral deletion and allows them to enter forbidden follicular and marginal zone niches. Immunity. 2004;20:785–798  Back to cited text no. 14
    
15.Segal JB, Powe NR. Prevalence of immune thrombocytopenia: analyses of administrative data. J Thromb Haemost. 2006;4:2377–2383  Back to cited text no. 15
    
16.Rodeghiero F, Stasi R, Gernsheimer T, Michel M, Provan D, Arnold DM, et al. Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood. 2009;113:2386–2393  Back to cited text no. 16
    
17.Zhu XJ, Shi Y, Peng J, Guo CS, Shan NN, Qin P, et al. The effects of BAFF and BAFF-R-Fc fusion protein in immune thrombocytopenia. Blood. 2009;114:5362–5367  Back to cited text no. 17
    
18.Wang CY, Zhu XJ, Hou M, Shi Y, Peng J, Sun JZ, et al. Effect of high-dose dexamethasone on BAFF and Tregs in patients with immune thrombocytopenic purpura. Zhonghua Xue Ye Xue Za Zhi. 2010;31:164–167  Back to cited text no. 18
    
19.Hase H, Kanno Y, Kojima M, Hasegawa K, Sakurai D, Kojima H, et al. BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex. Blood. 2004;103:2257–2265  Back to cited text no. 19
    
20.Aggarwal BB. Nuclear factor-κB: a transcription factor for all seasons. Expert Opin Ther Targets. 2007;11:109–110  Back to cited text no. 20
    
21.Espinosa G, Cervera R. Belimumab, a BLyS-specific inhibitor for the treatment of systemic lupus erythematosus. Drugs Today. 2010;46 (12):891–899  Back to cited text no. 21
    


    Figures

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

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



 

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