Kinetics of Protein-Nucleic Acid Interactions: Use of Salt Effects to Probe Mechanisms of Interactio

Abstract

Fc receptors (FcRs) are immunoglobulin-binding structures that enable antibodies to perform a variety of functions by forming connections between specific recognition and effector cells. Besides eliciting cytotoxicity, inducing secretion of mediators and endocytosis of opsonized particles, FcRs are involved in the regulation of antibody production, both as integral membrane proteins and as soluble molecules released from the cell surface. Most FcRs belong to the same family of proteins as their ligands (immunoglobulin superfamily). This review contains recent data obtained by use of monoclonal antibodies and cloning studies on FcRs and FcR-like molecules. The importance of fine specificity of receptor binding site(s) — that of the conformation of FcRs and their ligands in triggering signaling mechanisms — is analyzed. The regulatory function of membrane-bound and -released FcRs; the correlation between cell cycle, FcR expression, and release; as well as the possible mechanisms of these phenomena are discussed.     

Expression,characterization of recombinant human soluble BAFF secreted from CHO cell

B cell-activating factor of the TNF family (BAFF) is critical for B cell maturation and survival. Here, we constructed a stable CHO cell line, in which the expression level of soluble form of BAFF (sBAFF) was raised from 0.13 μg/ml to 0.55 μg/ml. Purified recombinant sBAFF from these CHO cells not only bound to its receptors but also co-stimulated the proliferation of human peripheral blood B lymphocyte in vitro. These results provided us with a useful basis for further studies about sBAFF-related research.     

转录因子对B细胞发育的调控作用

Ｂ细胞同其他血细胞一样,也是由多能造血干细胞发育为祖细胞,再发育成 末成熟细胞的。发育过程中,向Ｂ细胞发育的前 细胞逐渐推动向其他谱系发育的潜能,Ｂ细胞谱系特征性基因开始表达。近来的研究表明,Ｂ细胞发育过程中有多种转录因子参与,它们调节多种重要基因的转录,保证Ｂ细胞的正常发育。本文综述了与Ｂ细胞发育相关转录因子的最新研究进展。     

Interaction of plasminogen-related protein B with endothelial and smooth muscle cells in vitro

Plasminogen-related protein B (PRP-B) closely resembles the N-terminal plasminogen activation peptide, which is released from plasminogen during conversion to plasmin. We have previously demonstrated that the steady-state level of mRNA encoding PRP-B is increased within tumor tissues, and that recombinant PRP-B antagonizes neoplastic growth when administered systemically to mice harboring tumors, but no insights into the cell targets of PRP-B have been presented. Employing serum-free medium optimized for culturing human endothelial or smooth muscle cells, we show that recombinant PRP-B inhibits basic fibroblast growth factor-dependent cell migration for both cell types, as well as tube formation of endothelial cells. Comparison with the angiogenesis inhibitors angiostatin and endostatin revealed similar results. Recombinant PRP-B is effective in promoting cell attachment of endothelial and smooth muscle cells, and antibody interference experiments reveal that the interaction of recombinant PRP-B with endothelial cells is mediated at least in part by alpha(v)-containing integrins. Inhibition of angiogenesis in vivo by PRP-B was demonstrated in the chicken chorioallantoic membrane assay. PRP-B and other antiangiogenic molecules may elicit metabolic perturbations in endothelial cells as well as perivascular mesenchymal cells such as smooth muscle cells and pericytes.     

TAK1 regulates SCF expression to modulate PKBα activity that protects keratinocytes from ROS-induced apoptosis

Dysregulated reactive oxygen species (ROS) generation contributes to many human pathologies, including cancer and diabetes. During normal wound repair, inflammation-induced ROS production must be tightly controlled, but the mechanisms reining their generation remain unclear. Herein, we show that transforming growth factor β-activated kinase 1 (TAK1) directly regulates stem cell factor (SCF) expression, which activates the protein kinase B (PKB)α pro-survival pathway in a cell-autonomous manner to protect keratinocytes from ROS-mediated cell death. TAK1 is a pivotal inflammatory mediator whose expression was transiently elevated during wound healing, paralleling the ROS production profile. TAK1 deficiency in keratinocytes led to increased apoptosis in response to anoikis and TNF-α treatment and was associated with elevated ROS level as analyzed by FACS. Using organotypic skin co-culture and comparative growth factor array analysis, we revealed a cell-autonomous mechanism that involved the SCF/c-Kit/PKBα signaling cascade. Ectopic expression of TAK1 or treatment with exogenous recombinant SCF restored the increased ROS production and apoptotic cell death in TAK1-deficient keratinocytes. Conversely, normal keratinocytes treated with various inhibitors targeting the SCF/c-Kit/PKBα pathway exhibited increased ROS production and TNF-α- or anoikis-induced apoptosis. Our study reveals a novel anti-apoptotic role for SCF in keratinocytes and identifies TAK1 as a novel player uniting inflammation and ROS regulation in skin redox biology.     

The intracellular ratio of the membrane to the secreted form of immunoglobulin M heavy chain is a measure of the developmental stage of B cell lymphomas

Tumors of B lymphocyte origin have been used as models for normal B cells “frozen” at particular stages of their development. Surface properties, amount, and intracellular location of immunoglobulin and the synthesis of J chain have all been used as indicators of developmental stages. Each requires special techniques or yields data that are difficult to compare from one experiment to the next. For these reasons, we have developed a metric for B cell development that is simple to perform and allows quick quantitative comparisons of cell lines. It has recently been established that the membrane (μ_m) and secreted (μ_s) forms of the IgM heavy chain differ at their extreme carboxy termini. The two proteins differ slightly in size and are easily distinguished when they are compared without their carbohydrate on sodium dodecyl sulfate (SDS) polyacrylamide gels. We have examined four mouse tumors derived from the B lymphocyte lineage whose phenotypes resemble late pre-B cells (internal μ only; uninduced 70Z/3), small B lymphocytes (high levels of surface IgM; LPS-induced 70Z/3, WEHI 231), lymphoblasts (both membrane and secreted IgM; WEHI 279.1), and plasma cells (copious IgM secretion; MOPC 104E). Despite the fact the 70Z/3 and WEHI 231 secrete no detectable IgM, all of the tumors synthesize at least intracellular forms of both μ_m and μ_s. The proportion of μ_m is stable and is characteristic of each tumor. The 70Z/3 cells and WEHI 231 cells synthesize about 75% of their total μ as μ_m; WEHI 279.1 cells synthesize about 30% and MOPC 104E cells about 5% of their total μ as μ_m. The population of LPS-stimulated B lymphocytes shows a similar progression during its differentiation. The proportion of μ_m correlates with other developmentally regulated parameters (Fc receptor, Ia and plasma cell antigen levels, and J chain) and can be used as a simple metric for comparison with developing B lymphocytes and determination of the developmental stage of a B cell tumor.     

Pre-B cell colony enhancing factor (PBEF), a cytokine with multiple physiological functions

Pre-B cell colony enhancing factor (PBEF) is regarded as a proinflammatory cytokine. Named for its first discovered function as a pre-B cell colony enhancing factor, it has since been found to have many other functions relating to cell metabolism, inflammation, and immune modulation. It has also been found to have intracellular and extracellular forms, with the two overlapping in function. Most of the intracellular functions of PBEF are due to its role as a nicotinamide phosphoribosyltransferase (Nampt). It has been found in human endothelial cells, where it is able to induce angiogenesis through upregulation of VEGF and VEGFR and secretion of MCP-1. In human umbilical endothelial cells, PBEF increases levels of the protease MMP 2/9. PBEF has also been found in a variety of immune cells other than B cells and has been shown to inhibit apoptosis of macrophages. Extracellular PBEF has been shown to increase inflammatory cytokines, such as TNF-α, IL-1β, IL-16, and TGF-β1, and the chemokine receptor CCR3. PBEF also increases the production of IL-6, TNF-α, and IL-1β in CD14⁺ monocyctes, macrophages, and dendritic cells, enhances the effectiveness of T cells, and is vital to the development of both B and T lymphocytes. The purpose of this review is to summarize the recent advances in PBEF research.     

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Abstract Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig⁺ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.     

Activation—induced cell death in B lymphocytes

Upon encountering the antigen(Ag),the immune system can either develop a specific immune response of enter a specific state of unresponsiveness,tolerance.The response of B cells to their specific Ag can be activation and proliferation,leading to the immune response,or anergy and activation-induced cell death(AICD),leading to tolerance.AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR).BCR engagement initiates several signaling events such as activation of PLCγ,Ras,and PI3K,which generally speaking,lead to survival.However,in the absence of survival signals(CD40 or IL-4R engagement),BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases,expression of pro-apoptotic genes,and inhibition of pro-survival genes.The complex interplay between survival and death signals determines the B cell fate and, consequently,the immune response.     

神经生长因子对PC12细胞脂多糖损伤的保护作用及其机制

目的：细胞水平研究神经生长因子（NGF）对大鼠嗜铬细胞瘤细胞株（PC12细胞）脂多糖（LPS）损伤后的保护作用以及核转录因子（NF-κB）的活性影响,探讨药物作用机制。方法：PC12细胞常规培养后,建立LPS损伤模型,随后MTT观察不同浓度的LPS对PC12细胞损伤及NGF对LPS损伤的保护作用,同时用倒置显微镜和荧光显微镜下观察细胞状态,最后RT-PCR检测NF-κB的含量。结果：①PC12细胞LPS损伤有浓度梯度,随着LPS浓度的增加,PC12细胞的存活率不断下降;LPS损伤的同时加入不同浓度NGF,LPS损伤均有明显的改善。②显微镜观察显示PC12细胞形态学上的改变,表明NGF对LPS损伤有保护作用。③RT-PCR结果显示,LPS损伤细胞的NF-κB的相对表达量明显高于正常对照细胞,而药物治疗组的NF-κB表达量则接近于正常细胞。结论：目前,神经生长因子在脑内炎症后的细胞修复作用报道甚少,而本实验研究神经生长因子对PC12细胞LPS损伤起到保护作用,尤其是损伤后再修复作用,且其作用机制可能与NF-κB信号通路的调控有关。     

Regulation of Adhesion and Migration in the Germinal Center Microenvironment

T cell dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell homing to the GC and interaction with FDC critically depend on integrin-mediated adhesion. We have recently indentified the c-met-encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signalling pathway regulating B cell adhesion (van der Voort et al., 1997, J. Exp. Med. 185, 2121–2131). The c-Met protein is expressed on B cells localized in the dark zone of the GC (centroblasts) and is induced by CD40 plus BCR ligation. Stimulation of c-Met with HGF/SF. which is produced at high levels by tonsillar stromal cells and FDC, leads to receptor phosphorylation and to enhanced integrin-mediated adhesion of B cells to both VCAM-l and fibronectin. Interestingly, these responses to HGF/SF are promoted by heparan-sulfate proteoglycan forms of CD44 (CD44-HS). Like c-Met, CD44-HS is induced on B cells by CD40 ligation. It efficiently binds HGF/SF and strongly promotes signalling through c-Met. We conclude that integrin regulation during antigen specific B cell differentiation involves cross-talk between the HGF/SF-c-Met pathway and CD44-HS.     

Extracellular proteinase inhibitor-accelerated apoptosis is associated with B cell activating factor in mammary epithelial cells

The expression of extracellular proteinase inhibitor (Expi) gene was induced during the involution of mammary gland, when apoptosis occurs in this tissue. Transient transfection of Expi gene partially induced apoptosis of mammary epithelial HC11 cells. We developed the stable cell lines overexpressing Expi gene and found that overexpression of Expi accelerated apoptosis of mammary epithelial cells under serum starvation. To understand apoptosis pathway involved in the Expi overexpression, we examined the gene expression profile by using apoptosis gene array containing 243 genes. The subsequent confirmation of the altered gene expression by northern analysis demonstrated that overexpression of the Expi gene induced expression of several genes, which included B cell activating factor (BAFF), Bax, cytochrome c, caspase-9, caspase-3, caspase-6, and CIDE-A. From this study, we first demonstrate that BAFF is involved in mammary apoptosis. Furthermore, we have found that the Expi-accelerated apoptosis is mediated via BAFF receptor among three known BAFF receptors: BAFF receptor, tumor necrosis factor (TNF) receptor homologue TACI (transmembrane activator and CAML-interactor), and BCMA (another TNFR homologue, B cell maturation antigen). Our studies also demonstrate that the use of apoptosis array provides an efficient tool to identify apoptosis pathway involved in gene transfection.     

Depletion of B cells rejuvenates the peripheral B‐cell compartment but is insufficient to restore immune competence in aging

Aging is associated with increasing prevalence and severity of infections caused by a decline in bone marrow (BM) lymphopoiesis and reduced B‐cell repertoire diversity. The current study proposes a strategy to enhance immune responsiveness in aged mice and humans, through rejuvenation of the B lineage upon B‐cell depletion. We used hCD20Tg mice to deplete peripheral B cells in old and young mice, analyzing B‐cell subsets, repertoire and cellular functions in vitro, and immune responsiveness in vivo. Additionally, elderly patients, previously treated with rituximab healthy elderly and young individuals, were vaccinated against hepatitis B (HBV) after undergoing a detailed analysis for B‐cell compartments. B‐cell depletion in old mice resulted in rejuvenated B‐cell population that was derived from de novo synthesis in the bone marrow. The rejuvenated B cells exhibited a "young"‐like repertoire and cellular responsiveness to immune stimuli in vitro. Yet, mice treated with B‐cell depletion did not mount enhanced antibody responses to immunization in vivo, nor did they survive longer than control mice in "dirty" environment. Consistent with these results, peripheral B cells from elderly depleted patients showed a "young"‐like repertoire, population dynamics, and cellular responsiveness to stimulus. Nevertheless, the response rate to HBV vaccination was similar between elderly depleted and nondepleted subjects, although antibody titers were higher in depleted patients. This study proposes a proof of principle to rejuvenate the peripheral B‐cell compartment in aging, through B‐cell depletion. Further studies are warranted in order to apply this approach for enhancing humoral immune responsiveness among the elderly population.     

Development of tibulizumab,a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease

ABSTRACT

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.     

Serum B-cell activating factor predicts prognosis in nasal type,extranodal NK/T cell lymphoma

B-cell activating factor (BAFF) plays important roles in a variety of lymphoid malignancies. Compared with healthy adults, patients with non-Hodgkin lymphoma had higher level of serum BAFF, and it corresponded with disease severity, response for therapy and clinical outcome. Latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus (EBV) which is a known agent of nasal, extranodal NK/T cell lymphoma (ENKTCL) can switch the BAFF activating promoter leading to higher expression of BAFF in EBV-related tumor cells. However, the relationship between BAFF and ENKTCL has not been reported. Here we proposed a hypothesis that BAFF might play a regulatory role in ENKTCL development and maintenance. Our results showed that serum BAFF in ENKTCL patients was significantly higher than that in control group and negatively correlated with patients’ survival. It may be a valuable prognostic factor and deserved further study.     

Molecular basis for proline- and arginine-rich peptide inhibition of proteasome

PR39, a naturally occurring and cell-permeable proline- and arginine-rich peptide, blocks the degradation of inhibitor of nuclear factor κB (IκBα), thereby attenuating inflammation. It is a noncompetitive and reversible inhibitor of 20S proteasome. To identify its basis of action, we used solution NMR spectroscopy and mutational analyses of the active fragment, PR11, which identified amino acids required for human 20S proteasome inhibiting activity. We then examined PR11-mediated changes in the expression of nuclear factor κB-dependent genes in situ. The results provide prerequisites for proteasome inhibition by proline- and arginine-rich peptides, providing a powerful new tool to investigate inflammatory processes. These findings offer new leads in developing drugs to treat heart diseases or stroke.