Molecular structure, expression, and function analysis of BAFF gene in Chinese sucker, Myxocyprinus asiaticus.

B cell activating factor (BAFF), belonging to the tumor necrosis factor superfamily (TNFSF), is a critical cytokine for B cell survival and immunoglobulin secretion. Here, the BAFF gene of Chinese sucker (Myxocyprinus asiaticus) (MaBAFF) was cloned using RT-PCR and RACE (rapid amplification of cDNA end) techniques. The open reading frame (ORF) of MaBAFF encodes a 272-amino acid protein containing a transmembrane domain, a TNF family signature, and a putative furin protease cleavage site as seen in BAFFs from other species. Tissue expression profiles of MaBAFF determined by absolute and relative quantification of real-time quantitative PCR (qPCR) showed that MaBAFF is widely distributed in various tissues, with the highest expression in spleen. MaBAFF can be detected during fertilized egg period by RT-PCR. Upon induction by A. hydrophila, the expression of MaBAFF was up-regulated in spleen from 48 to 72 h, and the expression of BAFF and IgM all reached a peak at 48 h in head kidney. The soluble BAFF gene (MasBAFF) had been cloned into pET30a. SDS-PAGE and Western blotting analysis confirmed that the His-MasBAFF was efficiently expressed in Escherichia coli Rosset (DE3). CCK-8 assay indicated that the MasBAFF recombinant protein (200 ng/ml) could prolong the survival of peripheral blood leukocytes. Based on ELISA screening and Western blotting, monoclonal antibody 1-F2A3 against recombinant MasBAFF was selected and used for immunohistochemistry, which showed that BAFF-positive cells were detected in spleen and head kidney. Our results raise the possibility that MaBAFF may be useful to enhance immune efficacy in Chinese sucker disease defense.

Molecular expression analysis and characterization of rockfish (Sebastes schlegelii) B cell activating factor.

B cell activating factor (BAFF) is recognized as a member of the TNF superfamily proteins that mediate the immune responses. In this study, BAFF from rockfish (Sebastes schlegelii) (SsBAFF) was characterized based on its functional aspects. The open reading frame of SsBAFF is 804 bp in length and encodes a 267 long amino acid residue protein with predicted molecular weight of 29.48 kDa. The deduced protein sequence comprises with transmembrane domain, furin cleavage site and TNF domain carrying Flap binding site that unique to TNF family. Recombinant SsBAFF (rSsBAFF) significantly enhanced rockfish lymphocytes proliferation and viability in a concentration dependent-manner according to the results from water soluble tetrazolium salt (WST-1) assay and flow cytometric assay. rSsBAFF also modulated the expression of genes involved in anti-inflammatory (IL-10 and NFκB-2) and anti-apoptotic (Bcl-2 and Bax) signal pathways. SsBAFF mRNA expression was detected ubiquitously in all analyzed rockfish tissues, with the highest levels in the spleen and head kidney. Further, the expression of SsBAFF in spleen were significantly induced following LPS, poly (I:C) and Streptococcus iniae challenges. These findings strongly suggest that SsBAFF might play an important role in rockfish immune system through regulating the inflammatory response and proliferation of immune cells.

Study on the Mechanism of Selective Interaction of BR3 and BCMA with BAFF and APRIL.

BACKGROUND: B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) can activate signaling pathways by binding to specific receptors. BR3 (BAFF receptor) shows a unique selectivity for BAFF ligand, while B-cell maturation antigen (BCMA) exhibits a stronger interaction between APRIL-BCMA rather than BAFF-BCMA interaction. OBJECTIVE: The combined domains were fused with IgG1 Fc to better understand which domain affects the selective interaction of the receptor with BAFF and APRIL. METHODS: Since BR3 and BCMA both contain cysteine-rich repeat domains (CRD) with DxL motif, the binding domains of BR3 and BCMA were segmented into two parts in this study. BR3-1 (CFDLLVRHGVAC) and BCMA-1 (YFDSLLHACIPC) contained the conservative DxL motif, while BR3-2 (GLLRTPRPKPA) and BCMA-2 (QLRCSSNTPPLT) were adjacent to the CRDs yet still joined with BR3-1 and BCMA-1. Affinity between all possible combinations was then tested. RESULTS: The affinity of BR3-1-BCMA-2-Fc and BR3-1-BR3-2-Fc for BAFF was higher than BCMA-1-BR3-2-Fc and BCMA-1-BCMA-2-Fc. Moreover, BR3-1-BCMA-2-Fc and BCMA-1-BCMA- 2-Fc had affinity for APRIL, while BR3-1-BR3-2-Fc and BCMA-1-BR3-2-Fc hardly interacted with APRIL. CONCLUSION: BR3-1 region played a key role for interaction with BAFF, while BCMA-1 region exhibited weaker binding with BAFF. BCMA-2 region having an α-helix might contribute towards selectivity of APRIL-BCMA binding and BR3-2 rigid region had deleterious effects on the APRIL-BR3 interaction. These results provide comprehensive insights of the mechanism of selective interactions, and may promote specific antagonist design in the future.

A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors.

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.

Selective Binding BAFF/APRIL by the In and Outside Conservative Region of BCMA.

BACKGROUND: BAFF and APRIL are members of TNF superfamily. They play vital roles in the pathogenesis of autoimmune diseases. BCMA, a receptor, shows higher affinity for APRIL than for BAFF. Previous studies found that ligand binding specificity of BCMA may be determined by sequence outside DxL motif. OBJECTIVE: Investigate the contribution of a segment outside the DxL motif of BCMA for binding with ligands. METHOD: In this study, the conservative region of BCMA was divided into two segments: BCMA1 (NEYFDSLLHACIPC), a segment of the DXL motif and BCMA2 (QLRCSSNTPPLT), a segment outside of the DXL motif. Two peptides corresponding to the two segments were synthesized and their contribution to the ligands binding were detected by competitive ELISA. BCMA1-Fc fusion protein was also constructed, purified and analyzed by indirect and competitive ELISA. RESULTS: BCMA2 had no inhibiting effect on the interaction of BCMA-Fc and BCMA1-Fc with BAFF, but, it inhibited 22.5% and 15.2% of the interaction of BCMA-Fc and BCMA1-Fc with mAPRIL respectively. The binding rates of BCMA1-Fc for BAFF were 91.7%, but 80.6% for mAPRIL, suggesting that BCMA1-Fc without BCMA2, bound BAFF well and less efficiently to mAPRIL. CONCLUSION: These results suggest that BCMA2 outside of the conservative DxL motif of BCMA may play an important role in the binding selectivity to its ligands.

Unexpected Potency Differences between B-Cell-Activating Factor (BAFF) Antagonist Antibodies against Various Forms of BAFF: Trimer, 60-Mer, and Membrane-Bound.

Therapeutic agents antagonizing B-cell-activating factor/B-lymphocyte stimulator (BAFF/BLyS) are currently in clinical development for autoimmune diseases; belimumab is the first Food and Drug Administration-approved drug in more than 50 years for the treatment of lupus. As a member of the tumor necrosis factor superfamily, BAFF promotes B-cell survival and homeostasis and is overexpressed in patients with systemic lupus erythematosus and other autoimmune diseases. BAFF exists in three recognized forms: membrane-bound and two secreted, soluble forms of either trimeric or 60-mer oligomeric states. To date, most in vitro pharmacology studies of BAFF neglect one or more of these forms. Here, we report a comprehensive in vitro cell-based analysis of BAFF in assay systems that measure all forms of BAFF-mediated activation. We demonstrate the effects of these BAFF forms in both a primary human B-cell proliferation assay and in nuclear factor κB reporter assay systems in Chinese hamster ovary cells expressing BAFF receptors and transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI). In contrast to the mouse system, we find that BAFF trimer activates the human TACI receptor. Further, we profiled the activities of two clinically advanced BAFF antagonist antibodies, belimumab and tabalumab. Unexpectedly, we revealed differences in inhibitory potencies against the various BAFF forms, in particular that belimumab does not potently inhibit BAFF 60-mer. Through this increased understanding of the activity of BAFF antagonists against different forms of BAFF, we hope to influence the discovery of BAFF antagonist antibodies with distinct therapeutic mechanisms for improvement in the treatment of lupus or other related autoimmune pathologies.

Molecular structure, distribution, and immunology function of TNFSF13B (BAFF) in Nile tilapia (Oreochromis niloticus).

B cell-activating factor (BAFF)is a member of the tumor necrosis factor (TNF) family and plays roles in B cell survival and maturation. In this study, the full-length cDNA of Nile tilapia (Oreochromis niloticus) BAFF (tBAFF) was amplified from the spleen by reverse transcription PCR (RT-PCR). The open reading frame of this cDNA encodes a protein of 261 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFF. Real-time quantitative PCR (qPCR) analysis revealed that tBAFF is present in various tissues and is predominantly expressed in the spleen. The predicted three-dimensional (3D) structure of the Nile tilapia (Oreochromis niloticus) soluble BAFF (tsBAFF) monomer was determined by (3D) structure modeling monomeranalyzed by (3D) structure mouse counterpart. Both tsBAFF and EGFP/tsBAFF were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by SDS-PAGE and Western blot analysis. After purification, the EGFP/tsBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Laser scanning confocal microscopy showed that EGFP/tsBAFF bound to its receptor. In vitro, tsBAFF promoted the proliferation of Nile tilapia and mouse splenic B cells together with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, tsBAFF showed a similar proliferation-stimulating effect on mouse B cells compared to msBAFF. These findings indicate that tsBAFF plays an important role in the proliferation of Nile tilapia B cells and has functional cross-reactivity among Nile tilapia and mammals. Therefore, BAFF may represent a useful factor for enhancing immunological efficacy in animals.

A Comparison of Biological Activity of B Lymphocyte Stimulator (BLyS) Antagonist Peptibodies and the Elucidation of Possible BLyS Binding Sites.

B lymphocyte stimulator (BLyS) overexpression is associated with autoimmune diseases such as rheumatoid arthritis and lupus. BLyS antagonists are new effective therapeutic strategies that have been studied extensively. BLyS-binding peptides, BC originated from computer-aided drug design (CADD), 814 selected from the phage display library, as well as the 3-copy of BC (3-BC), were fused with human IgG1 Fc to constitute peptide-Fc fusion proteins, referred as peptibodies. BP-Fc, a peptibody possessing the identical sequence as BC-Fc but a His tag, was also constructed. The biological activities of these peptibodies were assessed by Enzyme-Linked Immuno Sorbent Assay (ELISA). Furthermore, the potential interacting orientations of BP and 814 with BLyS were studied. At 100 µg/ml, BC-Fc, BP-Fc, 814-Fc and 3-BC-Fc could distinctly inhibit 64 %, 50 %, 73 % and 56 % of the interaction of B cell maturation antigen (BCMA) with BLyS respectively. BP-Fc demonstrated 15 % higher binding ratio with BLyS than BC-Fc at 100 µg/ml. However, 814-Fc displayed at least 39 % higher BLyS-binding activity than BP-Fc at different concentrations. The binding capacity of 3-BC-Fc was slightly superior to BC-Fc. In addition, 814 and BP shared the identical domain on the surface of BLyS which involves in binding with BCMA, but owned the detached orientations. The discovery of possible locations of the BLyS-targeted peptides lays the foundation for the development of novel antagonists. Both BP-Fc and 3-BC-Fc fusion proteins could bind to BLyS in a dose-dependent manner and inhibit BLyS biological activity significantly, which might act as candidate agents for autoimmune disease therapy.

Characterisation of the TNF superfamily members CD40L and BAFF in the small-spotted catshark (Scyliorhinus canicula).

The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark (Scyliorhinus canicula). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies.

Molecular Mechanism of the Affinity Interactions between BAFF and Its Peptides by Molecular Simulations.

B-cell activating factor (BAFF) belonging to the TNF family, plays an important role in the proliferation and differentiation of B cells which renders it an attractive target for autoimmune diseases. Some peptides have been designed to target BAFF for the treatment of autoimmune diseases. Our previous studies suggested that peptides TA and DX-814 had competitive bioactivities compared to the natural peptide trans-membrane activator and calcium modulator and cyclophilin ligand interactor (TACI) in different binding orientations. In this study, we carried out molecular modeling and dynamics and molecular docking calculations to explore the structural and chemical features responsible for the binding affinities of these peptides. Binding free energy calculations, mutational analyses were also conducted to validate our findings. The result showed that hydrophobic and electrostatic interactions are the dominant forces for binding. DX-814 had a similar binding orientation with BCMA in a conserved hydrophobic pocket and formed electrostatic interaction with conserved arginine residues on the BAFF surface, compared with TA which might interact with a sub-pocket of BAFF in a different orientation. These results provide a thorough understanding of the binding mode between BAFF and its peptide inhibitors at the molecular level and further guide inhibitor design.

Molecular and functional characterization of BAFF from the Yangtze alligator (Alligator sinensis, Alligatoridae).

B-cell activating factor (BAFF) from the TNF family is critical for B-cell survival and maturation. In this study, we identified a Yangtze alligator (Alligator sinensis, Alligatoridae) BAFF cDNA, designated as asBAFF, using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The open reading frame of this cDNA encodes a 287-amino acid protein containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian and avian BAFF. The amino acid identity between biologically soluble asBAFF (assBAFF) and csBAFF, hsBAFF, and msBAFF is 94, 76, and 71%, respectively. Real-time quantitative PCR analysis showed that the asBAFF gene is strongly expressed in the spleen. Since BAFF is always expressed as inclusion bodies in bacteria, it is difficult to purify. To enhance the soluble expression of assBAFF in Escherichia coli, we fused the extracellular region of the asBAFF gene to a small ubiquitin-related modifier gene (SUMO). Purified assBAFF was able to promote the survival of splenic lymphocytes and co-stimulate the proliferation of mouse B cells with anti-mouse IgM. These findings suggest that asBAFF plays an important role in the survival and proliferation of Yangtze alligator B cells, and because it is evolutionarily highly conserved, functional cross-reactivity exists between mammalian and Yangtze alligator BAFF.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

In Silico Identification of Novel APRIL Peptide Antagonists and Binding Insights by Molecular Modeling and Immunosorbent Assays.

The "A proliferation inducing ligand" protein (APRIL) is a cytokine over-expressed in many transformed and tumoral cells acting onto two distinct receptors of the Tumoral Necrosis Factor B cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). We herein describe, through a detailed computational approach, the molecular interactions between TACI and its ligands APRIL and another structurally similar protein called B-cell activating factor (BAFF) by means of molecular dynamics. Dynamical analysis suggests R84 and D85 residues from TACI as possible mutation candidates, yielding increased affinity between TACI and APRIL. The association of computational simulations, site directed mutagenesis and peptide design could be a powerful tool, driving to better in vitro experiments. Our results contribute to the elucidation of APRIL signaling and help clarify the effects of blocking interaction between APRIL and its receptors through the use of particular peptides.

Comparative genomic of the BAFF and BAFF-like genes and immune response to bacteria of miiuy croaker (Miichthys miiuy).

As a member of the tumor necrosis factor (TNF) family, B cell activating factor (BAFF), also known as TNF ligand superfamily member 13B (TNF13B), playing a critical role in enhancing immune responses. BAFF is a central cytokine for B-cell survival, proliferation, maturation and immunoglobulin secretion. In the present study, we describe the identification of the miiuy croaker BAFF (designed MmBAFF) and BAFF-like (designed MmBAFF-like) genes. The cDNA of MmBAFF contains an open reading frame (ORF) of 795 nucleotides that are translated into a predicted 264 amino acids. The ORF of MmBAFF-like consists of 705 bases encoding 234 amino acids. Amino acid sequence comparison indicated that MmBAFF and MmBAFF-like possessed the TNF signatures, a predicted transmembrane domain, three conserved cysteine residues and a putative furin protease cleavage site, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the MmBAFF and MmBAFF-like monomer analyzed by comparative protein modeling revealed that they were very similar to human counterpart. Comparative genomic analysis revealed that the locations of MmBAFF and MmBAFF-like genes are conserved among the bony fish. Phylogenetic analysis shows the MmBAFF is most closely related to other teleost BAFFs with the highest similarity to Epinephelus awoara. And BAFF-like cluster get together first to BAFF cluster than three closely related TNF superfamily (TNFSF) members. Real-time quantitative PCR analysis shows the MmBAFF and MmBAFF-like transcripts are expressed in a wide range of tissues with the highest expression in skin and lymphoid tissue spleen. Upon induction by Vibrio anguillarum, their expressions are significantly upregulated in liver, spleen and kidney as compared to phosphate-buffered saline injected control fish. The association of increased BAFF expression after bacterial infection suggests that it plays a potentially important role in immune system of fish.

Equine adipose-derived stem cell (ASC) expresses BAFF and its receptors, which may be associated with the differentiation process of ASC towards adipocyte.

B cell activating factor (BAFF) and its receptors were regarded as elements of the immune system, regulating the fate of B cell. In recent years, these molecules were identified in a number of normal and pathological tissues, expanding their potential functions beyond the immune system. In this study, on the basis of molecular clone and prokaryotic expression of equine BAFF, we reported that equine adipose-derived stem cell (ASC) expressed BAFF and its receptors, which exhibited the increased expression during ASC adipogenic differentiation in vitro. Moreover, with the addition of recombinant protein His6-sBAFF, an increased differentiation of equine ASC towards adipocyte was detected. These results suggested that BAFF and its receptors might be associated with the differentiation process of ASC towards adipocyte in horse.

B-cell regulation and its application to transplantation.

There has been increasing interest in the role played by B cells and their associated antibody in the immune response to an allograft, driven by the need to undertake antibody-incompatible transplantation and evidence suggesting that B cells play a role in acute T-cell-mediated rejection and in acute and chronic antibody-mediated rejection. This review focuses on the molecular events, both activating and inhibitory, which control B-cell activation, and considers how this information might inform therapeutic strategies. Potential targets include the BAFF (B-cell-activating factor belonging to the tumour necrosis factor family) and CD40-CD40L pathways and inhibitory molecules, such as CD22 and FcγRIIB. B cells can also play an immunomodulatory role via interleukin (IL)10 production and may contribute to transplant tolerance. The expansion of allograft-specific IL10-producing B cells may be an additional therapeutic goal. Thus, the treatment paradigm required in transplantation has shifted from that of simple B-cell depletion, to that of a more subtle, differential manipulation of different B-cell subsets.

Characterization of the molecular structure, expression and bioactivity of the TNFSF13B (BAFF) gene of the South African clawed frog, Xenopus laevis.

B cell activating factor (BAFF), a member of the tumor necrosis factor family, is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF from the South African clawed frog (Xenopus laevis, designated xlBAFF) was cloned using rapid amplification of cDNA ends (RACE) techniques and RT-PCR. The full-length cDNA of xlBAFF consists of 1204 bases including an open reading frame (ORF) of 801 nucleotides that are translated into a predicted 266 amino acid protein. Sequence comparison indicated that the amino acids of xlBAFF possessed the TNF signature, including a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the xlBAFF monomer revealed that it was very similar to its counterparts. Real-time quantitative PCR analysis revealed that xlBAFF could be detected in various tissues and predominantly expressed in the spleen and other lymphoid tissue. The soluble xlBAFF had been cloned into a pET28a vector to express the recombinant protein. The His6-xlBAFF was efficiently expressed in Escherichia coli. BL21 (DE3) and its expressions were confirmed by SDS-PAGE and Western blotting analysis. After purification, laser scanning confocal microscopy analysis showed that xlBAFF could bind to its receptors on B cells. CCK-8 assays revealed that xlBAFF is not only able to promote survival/proliferation of South African clawed frog lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These results will allow for further investigation the use of X. laevis as an in vivo model for related studies.

Identification of TNF13b (BAFF) gene from grass carp (Ctenopharyngodon idella) and its immune response to bacteria and virus.

The TNF superfamily B cell activating factor (BAFF) is a central cytokine in several diseases. A BAFF gene has been cloned from grass carp (Ctenopharyngodon idella), analyzed its structure, and investigated its expression pattern in various tissues after Aeromonas hydrophila and Aquareovirus infection. The open reading frame of grass carp BAFF (gcBAFF) consists of 804 bases encoding 267 amino acids. Phylogenetic analysis shows the gcBAFF is most closely related to other teleost BAFFs with the highest similarity to zebrafish. RT-PCR analysis shows the gcBAFF transcript is expressed in a wide range of tissues with the highest expression in skin and spleen. Upon induction by A. hydrophila and Aquareovirus, its expression is significantly up-regulated in gill, liver, kidney, spleen and skin as compared to PBS injected fish. The association of increased BAFF expression after bacterial and viral infections suggests that it plays a potentially important role in immune system of fish.

BAFF/BLyS inhibitors: A new prospect for treatment of systemic lupus erythematosus.

In November 2009, Human Genome Sciences and Glaxo-Smith Kline [HGS (Rockville, Maryland) and GSK, respectively] announced that Belimumab, a neutralizing antibody to the tumour necrosis factor (TNF)-like ligand, B-cell activating factor (BAFF belonging to the TNF family, also named BLyS), met the primary endpoints in two phase III clinical trials in systemic lupus erythematosus (SLE, lupus). In March 2011, Belimumab was approved by the US Federal Drug Agency for treatment of SLE patients; this was followed in May with approval by the European Medicines Agency for use in the European Union. This is an exciting development as it is the first successful late-stage clinical trial in SLE in over 40 years. In the light of this breakthrough, we review the key data and research outcomes and examine how blocking BAFF in patients with SLE significantly improves clinical outcomes.

First BAFF gene cloned from an aquatic mammal.

The finless porpoise (Neophocaena phocaenoides) is one of the smallest cetacean species. Research into the immune system of the finless porpoise is essential to the protection of this species, but, to date, no genes coding for proteins from the tumor necrosis factor family (TNF family) have yet been reported from finless porpoises. The TNF B cell activating factor (BAFF) is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. It acts through its three receptors, BAFF-R, BCMA, and TACI. In the present study, the full-length cDNA of BAFF (designated NpBAFF) from the finless porpoise was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques, and its biological activities have been characterized. To our knowledge, this is the first report of any BAFF gene being cloned from an aquatic mammal. The full-length cDNA of NpBAFF consists of 1502 bases including an 852 bp open reading frame encoding 283 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known BAFF homologues. Sequence comparison indicated that the amino acid sequence of NpBAFF was very similar to its bovine (87.68%), porcine (76.33%), hircine (87.68%) and canine (82.19%) counterparts. The predicted three-dimensional (3D) structure of the NpsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its human counterpart. Phylogenetic analysis indicated that NpBAFF showed a notable homology with Artiodactyla BAFFs. The SUMO-NpsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that NpsBAFF could bind to its receptors on B cells. In vitro, MTT assays indicated that SUMO-NpsBAFF could promote the survival or proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that NpBAFF plays an important role in the survival or proliferation of B cells and has functional cross-reactivity among cetaceans and other mammals. The present findings may provide valuable information for research into the immune system of the finless porpoise.