Preliminary findings on the possible role of B-lymphocyte stimulator (BLyS) on diabetes-related periodontitis

Abstract The possible role of B-cell growth and differentiation-related cytokines on the pathogenesis of diabetes-related periodontitis has not been addressed so far. The aim of this study was to evaluate the effects of diabetes mellitus (DM) on the gene expression of proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), two major cytokines associated to survival, differentiation and maturation of B cells in biopsies from gingival tissue with periodontitis. Gingival biopsies were obtained from subjects with periodontitis (n = 17), with periodontitis and DM (n = 19) as well as from periodontally and systemically healthy controls (n = 10). Gene expressions for APRIL, BLyS, RANKL, OPG, TRAP and DC-STAMP were evaluated using qPCR. The expressions APRIL, BLyS, RANKL, OPG, TRAP and DC-STAMP were all higher in both periodontitis groups when compared to the control group (p < 0.05). Furthermore, the expressions of BLyS, TRAP and RANKL were significantly higher in the subjects with periodontitis and DM when compared to those with periodontitis alone (p < 0.05). The mRNA levels of BLyS correlated positively with RANKL in the subjects with periodontitis and DM (p < 0.05). BLyS is overexpressed in periodontitis tissues of subjects with type 2 DM, suggesting a possible role of this cytokine on the pathogenesis DM-related periodontitis.

Association Study of a Proliferation-inducing Ligand, Spermatogenesis Associated 8, Platelet-derived Growth Factor Receptor-alpha, and POLB Polymorphisms with Systemic Lupus Erythematosus in Chinese Han Population / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with complex genetic inheritance. This study was conducted to examine whether the association of a proliferation-inducing ligand (APRIL), spermatogenesis associated 8 (SPATA8), platelet-derived growth factor receptor-alpha (PDGFRA), and DNA polymerase beta (POLB) with SLE can be replicated in a Chinese Han population.</p><p><b>METHODS</b>Chinese SLE patients (n = 1247) and ethnically and geographically matched healthy controls (n = 1440) were genotyped for the APRIL, SPATA8, PDGFRA, and POLB single-nucleotide polymorphisms (SNPs), rs3803800, rs8023715, rs1364989, and rs12678588 using the Sequenom MassARRAY System.</p><p><b>RESULTS</b>The Chinese Han SLE patients and controls had statistically similar frequencies of alleles and genotypes of four gene polymorphisms. Moreover, no association signal was detected on different genetic models (additive, dominant, and recessive, all, P> 0.05) or in SLE subgroups stratified by various clinical manifestations (all, P> 0.05).</p><p><b>CONCLUSIONS</b>Different genetic backgrounds from different ancestries and various populations may result in different genetic risk factors for SLE. We did not detect any significant association with SNPs of APRIL, SPATA8, PDGFRA, and POLB.</p>

A proliferation-inducing ligand expression in breast cancer and its relationship with prognosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the relationship between a proliferation-inducing ligand (APRIL) expression in primary tumor foci of breast cancer and the patients' prognosis.</p><p><b>METHODS</b>Paraffin sections of surgical specimens were retrospectively collected from 130 stage I-III breast cancer patients who received surgery between January 2000 and December 2002 in our hospital. Immunohistochemistry was used to assess APRIL expression intensity in the tumor cells and density of interstitial APRIL-positive cells, and their association was analyzed with the density of interstitial CD4⁺ and CD8⁺ cells and with the histopathologic features, overall survival (OS), and disease-free survival (DFS) of the patients.</p><p><b>RESULTS</b>APRIL positive staining was found in the cytoplasm of the tumor cells, interstitial cells, and the extracellular matrix. APRIL intensity in the tumor cells was positively correlated with the density of interstitial APRIL-positive cells (P=0.009) and Ki67 (P=0.003). The density of interstitial APRIL-positive cells was positively correlated with the density of interstitial CD4⁺ cells (P<0.001) and CD8⁺ cells (P<0.001). In hormone receptor negative patients (ER- and PR-), multivariate COX regression identified the density of interstitial APRIL-positive cells as a positive prognostic factor for DFS (HR=0.313, 95% CI=0.107-0.920, P=0.035). CONCLUSIONSl APRIL is widely expressed in the interstitial immune cells in breast cancer. APRIL staining intensity in the tumor cells is positively correlated with tumor proliferation, indicating that the immune cells might promote tumor proliferation by secreting APRIL. A greater density of interstitial APRIL-positive cells is associated with a good prognosis in hormone receptor-negative patients.</p>

Progress in the study of BLyS and APRIL on regulating T cell responses in rheumatoid arthritis / 药学学报

B lymphocyte stimulator (BLyS), a tumor neurosis factor ligand superfamily, is an important factor of B cell survival and activation. However, BLyS also regulates T cell activation and survival, playing key roles in T cell-mediated autoimmune disorders. In the paper, we introduced the mechanisms of BLyS and a proliferation-inducing ligand (APRIL) regulating T cell responses and their roles in rheumatoid arthritis (RA).

Abnormal expression of APRIL in colorectal cancer cells promotes tumor growth and metastasis / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the effects of a proliferation-inducing ligand (APRlL) on colorectal cancer (CRC) cell growth and migration, and to observe the role of APRIL in CRC biological behavior.</p><p><b>METHODS</b>The siRNA plasmid vector targeting APRIL gene (APRIL-siRNA) was transfected into human colorectal cancer SW480 cells and recombinant human APRIL (rhAPRIL) was used to stimulate human colorectal cancer HCT-116 cells. Cell proliferation activity was analyzed using cell counting kit-8 (CCK-8), cell cycle was detected by flow cytometry, and the protein expression of cyclin D1, p21 and Bcl-2 was detected by Western blot analysis. Tumor cell migration and invasion were measured by Transwell chambers. RT-PCR was applied to examine the mRNA expression level of MMP-2 and MMP-9. APRIL-siRNA was used to transfect directly SW480 cells, which were injected subcutaneously into nude mice, then the tumor growth and metastasis were observed.</p><p><b>RESULTS</b>Cell proliferation ability of APRIL-siRNA-transfected SW480 cells was drastically repressed, and the percentage of G0/G1 phase cells was significantly increased (t = 4.12, P < 0.05), accompanied with depressed cyclin D1, Bcl-2 expression and elevated p21 expression. Cell proliferation ability of rhAPRIL-stimulated HCT-116 cells was promoted with a decreased G0/G1 phase ratio (t = 3.31, P < 0.05). cyclin D1 and Bcl-2 protein expression was up-regulated while p21 was down-regulated by rhAPRIL stimulation. Metastatic and invasive capacities of APRIL-siRNA-transfected SW480 cells were significantly inhibited compared with their respective controls (both P < 0.05), accompanied with the deregulated MMP-2 and MMP-9 mRNA expression. Metastatic and invasive capacities of rhAPRIL-stimulated HCT-116 cells were promoted with up-regulated MMP-2 and MMP-9 mRNA expression(both P < 0.05). Tumor growth in the group transfected with APRIL-siRNA appeared to be slower than that in the control groups and the expression of MMP-2, MMP-9 in tumor tissues was depressed in the APRIL-siRNA group.</p><p><b>CONCLUSIONS</b>APRIL facilitates tumor growth and metastasis, and is associated with carcinogenesis and prognosis. Our findings suggest that APRIL might be used as a novel target for the intervention and therapy of colorectal cancer.</p>

Preparation of polyclonal antibody against sAPRIL and analysis of function in suppressing sAPRIL-mediated lymphocyte proliferation / 中国实验血液学杂志

This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p < 0.05). It is concluded the polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.

Effect of BAFF/APRIL mRNA expression induced by glucocorticoid and bortezomib in multiple myeloma cells in vitro / 中国实验血液学杂志

The study was purposed to detect BAFF/APRIL gene expression changes in bone marrow mononuclear cells (BMMNC) and myeloma cell line U266 after interference with glucocorticoid and bortezomib. After separation of BMMNC from 7 patients with multiple myeloma, BAFF/APRIL mRNA expression in BMMNC and U266 cell line was detected by real-time PCR after treated with dexamethasone 100, 200 µg/ml, methylprednisolone 100, 200 µg/ml, bortezomib 0.1 µg/ml alone and dexamethasone or methylprednisolone combined with bortezomib respectively for 48 hours. The results showed that U266 cells and BMMNC of untreated MM patients highly expressed BAFF/APRIL genes. When dexamethasone, methylprednisolone or bortezomib was added to U266 cells or BMMNC alone, BAFF/APRIL gene expression decreased as compared with the blank control (p < 0.01). The inhibiting effect of bortezomib to BAFF/APRIL expression was obviously strong(p < 0.05). When dexamethasone or methylprednisolone combined with bortezomib, the BAFF/APRIL gene expression further decreased compared with dexamethasone or methylprednisolone alone (p < 0.01). As compared with the group of methylprednisolone combined with bortezomib, BAFF/APRIL gene expression decreased in dexamethasone combined with bortezomib with a statistically significant difference (p < 0.05). It is concluded that the expression of BAFF/APRIL gene is down-regulated after bing treated with glucocorticoids and bortezomib, which suggests that besides the glucocorticoid receptor and proteasomes targets, BAFF/APRIL and their receptor sites may be new targets of glucocorticoids and bortezomib.

Lentivirus-mediated RNA interference suppresses APRIL expression and enhances chemosensitivity in colorectal cancer cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of lentivirus-mediated RNA interference (RNAi) targeting a proliferation-inducing ligand (APRIL) on the chemosensitivity to 5-FU of colorectal cancer cell line LoVo.</p><p><b>METHODS</b>The lentiviral vector siRNA-APRIL was constructed and verified by PCR and DNA sequencing. LoVo cells were transfected with siRNA-APRIL plasmid, non-targeting siRNA plasmid, or empty plasmid. Forty-eight hours after the transfection, the cells were examined for APRIL expression using Western blot. Seventy-two hours after treatment with 10 µg/ml 5-FU, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate following 5-FU exposure was detected by MTT assay.</p><p><b>RESULTS</b>PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting APRIL gene was successfully inserted into the lentiviral vector. siRNA-APRIL transfection resulted in obviously reduced expression of APRIL in LoVo cells. After 5-FU exposure, the apoptosis rate of siRNA-APRIL-transfected cells were increased to (21.12∓3.35)%, significantly higher than that in cells transfected with the non-targeting plasmid or the empty plasmid [(13.06∓1.92)% and (12.28∓1.79)%, respectively, P<0.01]; the cell number in G0/G1 phase increased while that in G2/M phase decreased in siRNA-APRIL-transfected cells. The growth inhibition rate in siRNA-APRIL group was (59.67∓5.03)%, significantly higher than that in the other two groups [(42.33∓4.16)% and (39.67∓4.73)%, respectively, P<0.01].</p><p><b>CONCLUSION</b>Lentivirus-mediated RNAi targeting APRIL can effectively suppress the expression of APRIL in LoVo cells and enhance the chemosensitivity of the cells to 5-FU.</p>

Effect of APRIL on growth and apoptosis in transplanted tumor with human colorectal cancer cell line SW480 in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To study the effect of pGCsi-H1-APRIL on the growth of human colorectal cancer cells in transplated tumor in nude mice and to improve the effect of APRIL on proliferation and apoptosis of colorectal cancer (CRC).</p><p><b>METHODS</b>Human CRC model was established in nude mice, and the nude mice were treated with APRIL siRNA twice per week for 2 weeks. APRIL mRNA expression was surveyed by PCR and APRIL protein expression was detected by immunohistochemistry. The expression of PCNA protein was detected by ELISA. The expression of bcl-2 and bcl-xl was assessed by immunohistochemical staining, and TUNEL staining was used to detect apoptosis.</p><p><b>RESULTS</b>The expression of APRIL mRNA in the APRIL siRNA group was (0.13 ± 0.05) × 10(-3), significantly lower than that in the vector group (0.95 ± 0.04) × 10(-3) and the PBS group (0.96 ± 0.05) × 10(-3). The expression of APRIL protein in the APRIL siRNA group was (87.5 ± 5.0)% lower than that in the vector and PBS groups (P < 0.05). APRIL siRNA significantly suppressed the growth of SW480 tumor: the IR (inhibitory rate) of APRIL siRNA group was (60.7 ± 1.5)% (P < 0.05). The expression of PCNA in APRIL siRNA group was (176.8 ± 18.1) ng/ml, was (56.5 ± 2.0)% lower than that of PBS group (328.4 ± 22.8) ng/ml. Furthermore, the expressions of anti-apoptosis proteins bcl-2 and bcl-xl of APRIL siRNA group were (82.6 ± 4.5)% and (79.2 ± 3.5)% lower than those of the PBS group. The apoptotic rate of the APRIL siRNA group was 40.1% ± 2.5%, significantly higher than that in the vector group (2.5 ± 0.1)% and PBS group (2.5 ± 0.2)% (P < 0.05).</p><p><b>CONCLUSION</b>APRIL siRNA may significantly suppress the growth and promote apoptosis in transplanted tumor of human colorectal cancer in nude mice. APRIL may become a candidate gene of gene therapy of human colorectal cancer.</p>

april mRNA expression in newly diagnosed leukemia patients / 中国实验血液学杂志

This study was aimed to quantitatively detect the levels of april mRNA expression in leukemia patients so as to provide theoretical basis for the target therapy directing at april in leukemia. Real time fluorescent quantitative PCR was used to detect the relative expression level of april mRNA in newly diagnosed leukemia patients and to analyze the changes of its expression level in various type of leukemia. The results showed that the april mRNA expression level in acute leukemia (AL) patients was significantly higher than that in normal controls, there was statistical difference between them (p < 0.05); april mRNA expression level in acute myeloid leukemia (AML) patients was significantly higher than that in normal controls (p < 0.05) and positively correlated with white blood cell count ≥ 20.0 × 10(9)/L (p < 0.05), but not related with extramedullary infiltration and the expression of CD34. Except for acute promyelocytic leukemia (APL), april mRNA expression level was negatively correlated with sensitivity of patients to chemotherapy. april mRNA expression levels in acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML) patients were not higher than that in normal controls, there was no statistical difference between them (p > 0.05). It is concluded that april gene overexpression exits in AML patients. APRIL protein produced by AML cells probably plays an important role in abnormal proliferation and drug-resistance of AML cells.

Expression and significance of BLyS/April in patients with acute idiopathic thrombocytopenic purpura / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the roles of B-lymphocyte stimulator/ a proliferation-inducing ligand (BLyS/April) in immunological pathogenesis of idiopathic thrombocytopenic purpura (ITP).</p><p><b>METHODS</b>Thirty ITP children and 30 age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the mRNA levels of BLyS/ April, receptors for BLyS/April (BR3, BCMA and TACI) and cytokines in ITP patients. Flow cytometry was performed to measure relative mean fluorescence intensity (relative MFI) for platelet-associated immunoglobulin G (PAIgG).</p><p><b>RESULTS</b>(1) The transcription levels of BLyS/April in monocytes/macrophage [(8.30 +/- 2.31) x 10(-1) and (7.51 +/- 1.93) x 10(-3), respectively] were significantly up-regulated in acute ITP compared with that in healthy controls [(3.95 +/- 1.04) x 10(-1) and (3.08 +/- 0.82) x 10(-3), respectively] (P < 0.0.1). (2) Expression levels of the BLyS/April receptors BR3, BCMA and TACI mRNA were remarkably raised during acute phase of ITP (P < 0.01). (3) The mRNA levels of cytokines, including IL-4, IL-5, IL-6, IL-10 and IL-15, were significantly higher in acute phase ITP than in healthy controls (P < 0.01). (4) The mRNA levels of IL-10 and IFN-alpha were significantly elevated in acute phase of ITP. (5) Relative MFI of acute phase ITP patients (67.4 +/- 28.1) was higher than that in healthy controls (19.5 +/- 8.5) (P < 0.01), and there was a significant positive correlation between relative MFI and BLyS/April as well as their receptors (BR3, BCMA and TACI) (r = 0.56, 0.53, 0.62, 0.70, 0.45, respectively, P < 0.01), relative MFI in ITP patients decreased after treatment.</p><p><b>CONCLUSION</b>Over-expression of BLyS/April may be one of factors contributed to the immunological dysfunction in ITP.</p>

Cloning, soluble expression and characterization of human sBCMA / 生物工程学报

BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.