Abstract: OBJECTIVE: To determine whether serum levels of a proliferation-inducing ligand (APRIL) are altered in patients with systemic lupus erythematosus (SLE), and correlate with disease parameters. METHODS: Clinical and biological parameters were analyzed for 43 patients that fulfilled American College of Rheumatology (ACR) criteria for SLE classification and were positive for anti dsDNA antibodies at least once in their medical record. Tests included measurement of serum levels of the TNF family members APRIL and B lymphocyte stimulator (BLyS, a cytokine shown to promote SLE disease). RESULTS: Median APRIL levels were elevated in SLE patients compared to osteoarthritis patients and healthy controls, but did not correlate with the SLE Disease Activity Index (SLEDAI). APRIL serum levels showed an inverse correlation with BLyS serum levels (r = 0.339; p = 0.03). For SLE patients with positive anti-double-stranded DNA (anti-dsDNA) titers (> 40 UA/ml) at inclusion (n = 25), circulating APRIL was inversely correlated with BLyS levels (r = -0.465; p = 0.022) and anti-dsDNA antibody titers (r = -0.411; p = 0.046). In a follow-up study at their second visit, 27 patients showed an inverse correlation with BLyS (r = 0.398; p = 0.03) and anti-dsDNA (r = -0.408; p = 0.03) titers, as well as an inverse correlation of APRIL serum levels with SLEDAI (r = -0.408; p = 0.01). CONCLUSION: The inverse correlation observed between APRIL and BLyS suggests that APRIL acts as a protective factor. APRIL and BLyS may thus have opposite roles in SLE, which must be considered when defining therapeutic applications of these cytokines.

Abstract: APRIL (a proliferation-inducing ligand) and BLyS (B lymphocyte stimulator) expression have been reported in chronic lymphocytic leukemia (CLL) cells. We examined APRIL and BLyS serum levels in CLL patients and evaluated the prognostic significance of APRIL expression on survival.

Abstract: A proliferation inducing ligand (APRIL) and B cell activating factor belonging to the TNF family (BAFF/BLyS) have been implicated in IgA class switch recombination in thymus-independent (TI) B cell responses. Dendritic cells (DC) are thought to regulate Ig class switching in TI B cell responses by providing B cells with cytokines, including APRIL and BAFF. We therefore set out to analyze the regulation of APRIL and BAFF expression by human monocyte-derived DC (moDC). We observed that moDC produce and secrete APRIL, but could not detect expression of BAFF. Importantly, stimulation with the Toll-like receptor ligands CpG and poly I:C specifically induced APRIL production, while other Toll-like receptor ligands were ineffective. The increase in APRIL was dependent on translation, but surprisingly not transcription. Instead, enhanced APRIL production and secretion resulted from activation of protein kinase receptor (PKR), as it was completely inhibited by the specific inhibitor of PKR, 2-aminopurine. This suggests that the specific induction of APRIL by CpG and poly I:C, and the signal integration by PKR, are regulated by translational modification and hint at a role for APRIL in the TI B cell response to viral infections.

Abstract: APRIL, a proliferation-inducing ligand, is a member of the tumor necrosis factor (TNF) family that is expressed by various types of tumors and influences their growth in vitro and in vivo. Two receptors, transmembrane activator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), bind APRIL, but neither is essential for the tumor-promoting effects, suggesting that a third receptor exists. Here, we report that APRIL specifically binds to heparan sulfate proteoglycans (HSPG) on the surface of tumor cells. This binding is mediated by the heparin sulfate side chains and can be inhibited by heparin. Importantly, BCMA and HSPG do not compete, but can bind APRIL simultaneously, suggesting that different regions in APRIL are critical for either interaction. In agreement, mutation of three lysines in a putative heparin sulfate-binding motif, which is not part of the TNF fold, destroys interaction with HSPG, while binding to BCMA is unaffected. Finally, whereas interaction of APRIL with HSPG does not influence APRIL-induced proliferation of T cells, it is crucial for its tumor growth-promoting activities. We therefore conclude that either HSPG serve as a receptor for APRIL or that HSPG binding allows APRIL to interact with a receptor that promotes tumor growth.

Abstract: A tumor-supporting role for the TNF-like ligand APRIL has been suggested. Here we describe that 9- to 12-month-old APRIL transgenic mice develop lymphoid tumors that originate from expansion of the peritoneal B-1 B cell population. Aging APRIL transgenic mice develop progressive hyperplasia in mesenteric lymph nodes and Peyer's patches, disorganization of affected lymphoid tissues, mucosal and capsular infiltration, and eventual tumor cell infiltration into nonlymphoid tissues such as kidney and liver. We detected significantly increased APRIL levels in sera of B cell chronic lymphoid leukemia (B-CLL) patients, indicating that APRIL promotes onset of B-1-associated neoplasms and that APRIL antagonism may provide a therapeutic strategy to treat B-CLL patients.

Abstract: Differential aspects of the host immune response generated by Trypanosoma cruzi infection were examined in two different mouse strains, BALB/c (haplotype H2-Kd) which does not overcome the acute phase of the infection and C57BL/6 (haplotype H2-Kb) which survives to the acute phase. After infection an increase in CD3+ T cells was observed in both mouse strains in the peritoneal cavity. However, while the CD3+ T cells from the BALB/c mice showed an increase in the IL-4 cytokine expression level, the same type of cells from the C57BL/6 mice showed an increase in IFN-gamma expression. In addition, only the macrophages from the C57BL/6 mice were activated secreting IL-12 and TNF-alpha and producing, moreover, high levels of nitrites. It was observed that also after parasite infection the expression of macrophage and dendritic cells CD40 and CD86 co-stimulation molecules from the spleen were diminished in BALB/c but not in C57BL/6 mice. In correlation with this observation the macrophages from the spleen of infected BALB/c mice secreted lower concentrations of nitrites than the C57BL/6 mouse cells. Also, the spleen dendritic cells from infected BALB/c mice had a small potential to present alloantigens in contrast to that observed in the infected C57BL/6 mouse cells.

Abstract: We analyse the effect of Trypanosoma cruzi heat-shock protein-70 (HSP70) on the maturation of murine dendritic cells (DC)generated from bone marrow precursor cells. The results obtained show that HSP70, both alone and fused to the KMP11 antigen, as well as a HSP70 fragment, is capable of maturing murine DC. Mature DC have enhanced expression of IL12, TNF-alpha cytokines, costimulation molecules and activation markers, showing a clear increase in the allostimulatory capacity. These findings suggest that T. cruzi HSP70 may be a very useful vehicle for developing DC-based immunoprophylaxis and therapy against infections.

Abstract: Cytotoxic T lymphocyte response against Jurkat-A2/K(b) cells expressing the T. cruzi KMP11 protein has been evaluated after immunization of C57BL/6-A2/K(b) transgenic mice with the KMP11 and KMP11-HSP70 recombinant proteins. The results show that mice immunized with KMP11 covalently fused to the T. cruzi HSP70 protein, but not mice immunized with KMP11 alone, elicit a CTL response against the Jurkat-A2/K(b) cells expressing the KMP11 protein. The data also show that spleen cells from mice immunized with the fusion protein and stimulated with the K1 peptide induce lysis of both the Jurkat-A2/K(b) cells transfected with the KMP11 coding gene and the Jurkat-A2-K(b) cells pulsed with the K1 peptide. Splenocytes stimulated with the K3 peptide induce lysis of the Jurkat-A2/K(b) cells loaded with the K3 peptide but they do not recognize the target cells expressing the KMP11 protein. Similar results were obtained using lymph node from mice immunized with the peptides. Thus, we believe there are two cytotoxic T cell epitopes restricted to the A2 molecule (K1(KMP11) (4-12) and K3(KMP11) (41-50)) in the KMP11 protein, and suggest that the K1 peptide could be considered an immunodominant antigen whilst the K3 peptide may be regarded as a cryptic epitope. The fact that the CTL lines induced in B6-A2/K(b) mice recognize human cells expressing KMP11 protein, indicates that the KMP11 antigen fused to HSP70 could be a good candidate for the induction of immunoprotective cytotoxic responses against T. cruzi natural infection.

Abstract: Murine immunization with Trypanosoma cruzi KMP11-HSP70 fused genes but not the KMP11 gene alone elicited both an immunoglobulin G2a long-lasting humoral immune response against KMP11 protein and activation of CD8+ cytotoxic T lymphocytes specific for two KMP11 peptides containing A2 motifs. Moreover, protection against the parasite challenge was observed after immunization with the chimeric gene.

Abstract: We developed a method to highly purify CD4+ and CD8+ T cells from murine spleen by negative enrichment strategy. Single-cell suspensions of spleen cells were depleted from erythrocytes by ammonium chloride-mediated lysis. The obtained cell suspension contained approximately 28% CD4+ cells and 14% CD8+ cells. Passing of these cells over a nylon wool column removed up to 75% of all cells, leading to a suspension containing approximately 50% CD4+ and 23% CD8+ cells. These cells were further purified by a single immunomagnetic depletion step using a panel of eight antibodies in combination with MACS magnetic beads and an autoMACS machine. After purification, cells were viable and mostly non-activated based on the expression of activation markers and did not or only minimally respond to polyclonal stimuli such as soluble anti-CD3 antibodies or Concanavalin A. With this method, 19-38% of all CD4 cells and 10-29% of all CD8 cells in a spleen cell suspension were recovered at the mentioned purity. The whole procedure is fast (<4 h of preparation), simple and cost effective, as all antibodies and the magnetic beads have been titrated to the minimal concentration needed for purification. The method is highly reproducible, routinely leading to CD4+ cells with >97% purity (range 97.4-99%) and CD8+ cells with >96% purity (range 95.6-96.7%). The described protocol should facilitate studies aiming at the physiology of "untouched" murine T cells.