New roles for the BLyS/BAFF family in antigen-experienced B cell niches

BLyS family members govern selection and survival of cells in the pre-immune B cell compartment, and emerging evidence suggests similar roles in antigen-experienced B cell pools. We review the features of this family, with particular emphasis on recent findings of how BLyS influences affinity maturation in germinal centers, which lie at the intersection of the pre-immune and antigen-experienced B cell compartments. We propose a model whereby tolerogenic selection at the transitional stage and affinity maturation in the germinal center employ the same BLyS driven mechanism.     

Mechanism of action of aflatoxin B1 in Bacillus megaterium.

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

Cutting edge: B cell receptor signals regulate BLyS receptor levels in mature B cells and their immediate progenitors

This study examines how B lymphocyte stimulator (BLyS) receptor expression and responsiveness are influenced by B cell receptor (BcR) signaling. Our results show that resting and BcR-stimulated B cells are dependent on BLyS for survival and that B cells remain BLyS responsive during BcR-induced activation. Further, BcR ligation up-regulates expression of the BLySR B cell maturation defect/BLySR3 (Bcmd/BR3), but not other known BLySRs. Finally, the coupling of BcR signaling with Bcmd/BR3 expression is limited to late transitional and mature B cells. Together, these findings establish the coupling of BcR signaling with Bcmd/BR3 expression as a fundamental aspect of follicular B cell selection, survival, and activation.     

An optimized B lymphocyte stimulator (BLyS) antagonist peptide inhibits the interaction of BLyS with BCMA

B lymphocyte stimulator (BLyS) antagonists are new therapeutic reagents for treating the autoimmune diseases. Peptibodies can inhibit the bioactivity of BLyS, the same as other BLyS antagonists: decoyed BLyS receptors and anti-BLyS antibodies. In this study, a new optimized BLyS antagonist peptide was designed according to our previous work by the computer-aided homology modeling. Competitive ELISA showed that the peptide at 100 μg/ml could inhibit 54 % of the BCMA-Fc binding to BLyS. To maintain its stability and spatial conformation, the peptide was fused to human IgG1 Fc to form a peptide-Fc fusion protein—a novel peptibody by gene engineering. ELISA indicated that the peptibody could bind with BLyS in dosage-dependent manner as BCMA-Fc did. This study highlights the possibility of designing and optimizing BLyS antagonist peptides with high biopotency by the computer-aided design. Thus, these peptides could neutralize BLyS activity and be potential antagonists to treat autoimmune diseases related with BLyS overexpression.     

B淋巴细胞刺激因子（BLyS）研究进展

B淋巴细胞刺激因子（BLyS）是1999年新发现的一种重要的细胞因子,属于肿瘤坏死因子(TNF)超家族成员.在体液免疫调控中起重要作用.它能强烈地刺激B淋巴细胞的增殖和分化并分泌大量免疫球蛋白(主要为IgM);在体外其过量表达能促进多种B系肿瘤细胞的生长.BLyS转基因小鼠出现严重的红斑狼疮样症状.对BLyS的基因和蛋白质结构、免疫调控功能、受体和信号通路及其在自身免疫性疾病和恶性肿瘤中的作用进行了综述.     

Mechanism of action of cytochalasin B on actin

Substoichiometric cytochalasin B (CB) inhibits both the rate of actin polymerization and the interaction of actin filaments in solution. The polymerization rate is reduced by inhibition of actin monomer addition to the "barbed" end of the filaments where monomers normally add more rapidly. 2 microM CB reduces the polymerization rate by up to 90%, but has little effect on the rate of monomer addition at the slow ("pointed") end of the filaments and no effect on the rate of filament annealing. Under most ionic conditions tested, 2 microM CB reduces the steady state high shear viscosity by 10-20% and increases the steady state monomer concentration by a factor of 2.5 or less. In addition to the effects on the polymerization process, 2 microM CB strongly reduces the low shear viscosity of actin filaments alone and actin filaments cross-linked by a variety of macromolecules. This may be due to inhibition of actin filament-filament interactions which normally contribute to network formation. Since the inhibition of monomer addition and of actin filament network formation have approximately the same CB concentration dependence, a common CB binding site, probably the barbed end of the filament, may be responsible for both effects.     

Mechanism of action of aflatoxin B1 in Bacillus megaterium

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.     

Mechanism of uricase action.

Uricase (urate:oxygen oxidoreductase, EC 1.7.3.3) exposes a positional and steric specificity in the enzymic conversion of urate to allantoin. C-2 of urate was recovered as C-2 of allantoin. By the consecutive oxidation and hydrolysis reactions a levorotatory intermediate was formed, presumably (-)-2-oxo-4-hydroxy-4-carbohydroxy-5-ureido-imidazoline. The absorption and optical rotation dispersion spectra of the intermediate were established. In the presence of borate buffer, the intermediate was transformed to (+)-alloxanate. The decay of the former compound depends on general base and acid catalysis. RS-(+/-)-allantoin was formed by chemical decarboxylation and S-(+)-allantoin by enzymic decarboxylation.     

Mechanism of fluoroquinolone action.

When fluoroquinolones bind to gyrase or topoisomerase IV in the presence of DNA, they alter protein conformation. DNA cleavage results with diminished religation, so the enzymes are trapped in ternary complexes with drug and cleaved DNA. Preferential localization of gyrase ahead of replication forks and topoisomerase IV behind them causes fluoroquinolone-mediated complexes with the two enzymes to have different physiological consequences.     

B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels

Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and ΔBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and ΔBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and ΔBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and ΔBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.     

B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels

Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and DeltaBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and DeltaBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and DeltaBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and DeltaBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.     

B 淋巴细胞刺激因子（BLyS）调控相关蛋白的研究进展

B淋巴细胞刺激因子是肿瘤坏死因子超家族的成员之一,它同自身免疫疾病的发生发展密切相关,BLyS过度表达会促使B淋巴细胞增殖并分泌自身抗体。BLyS也是治疗自身免疫疾病的重要靶点。关于BLyS的调控涉及了许多蛋白。本文主要就G-CSF、TGF-β1、IFN-γ、IL-10和Poly(I:C)等对BLyS的调控作用作一综述。     

Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice

The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.     

The role of mast cell degranulation products in mast cell hyperplasia. I. Mechanism of action of nerve growth factor

A variety of mast cell degranulating agents have previously been shown to induce mast cell hyperplasia in adult rats. In neonates 2.5 S nerve growth factor (NGF) induces a hyperplasia of both mucosal and connective tissue mast cells (MMC and CTMC). We have examined the role of the potent mast cell degranulating properties of NGF on its ability to induce mast cell hyperplasia. Administration of NGF in combination with the mast cell stabilizing agent disodium cromoglycate was found to abrogate the CTMC hyperplasia induced by NGF alone. Treatment of neonatal rats with the alternate degranulating agent compound 48/80 was found to induce a limited CTMC but not a MMC hyperplasia. A supernatant obtained by degranulating purified adult rat peritoneal mast cells with anti-IgE was found to induce hyperplasia of the CTMC population similar to that observed with NGF administration. However, this degranulation product supernatant only induced a limited MMC hyperplasia as judged by RMCP II content of the tissues. These results suggest that NGF has dual action inducing mast cell hyperplasia; CTMC hyperplasia being dependent on the ability of NGF to degranulate mast cells. MMC hyperplasia induced by NGF is independent of CTMC degranulation. Degranulation products from peritoneal mast cells act to increase both MMC and CTMC populations in the neonate. These data suggest that the CTMC population may be regulated by an autocrine positive feedback mechanism in vivo.     

Mechanism of action of amylin in bone.

Amylin is a 37 amino acid peptide produced mainly by beta-cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene-related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT-like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]- induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly approximately 60-fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast-like multinucleated cells (MNCs) formed in co-cultures of mouse osteoblasts and bone marrow cells in the presence of 1 alpha,25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast-like MNCs, but only at 60-fold higher concentrations than human CT. Specific binding of [125I]-human CT to osteoclast-like MNCs was detected (dissociation constant, 3 x 10(-8) M; binding sites, 3 x 10(7) per cell). To displace the bound [125I]-human CT from osteoclast-like MNCs, about 170-fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]-amylin and [125I]-CGRP to osteoclast-like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast-like cells (KS-4) and in mouse primary osteoblast-like cells. Amylin was a weak agonist for cAMP production in KS-4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8-37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS-4 cells. Amylin, but not CT, displaced the bound [125I]-human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast-like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT greater than amylin not equal to CGRP in osteoclast-like MNCs and CGRP greater amylin much greater than CT in osteoblast-like cells.     

Mechanism of metolachlor action due to alterations in cell cycle progression

Metolachlor, a commonly used herbicide in the Midwestern USA, functions by inhibiting chlorophyll and protein synthesis in target plants. Herbicide exposure has led to detrimental effects in several organisms, affecting their growth and behavior; however, its mechanism of action in nontarget organisms is not yet clear. The EPA does not currently have enforceable regulations for maximal limits allowed in drinking water. Previous growth studies from our lab have demonstrated that increasing metolachlor concentrations and increasing time of exposure results in decreased growth of liver cells. The objective of this study was to elucidate a mechanism for this decrease of HepG2 cell growth after herbicide exposure. Results show that metolachlor at environmentally relevant levels (50–100 ppb) that previously led to decreased cell number does not lead to cell death by either necrosis or apoptosis. However, it was demonstrated that the levels of the retinoblastoma protein including two of its hyperphosphorylated forms are decreased in metolachlor exposed cells possibly leading to cell cycle arrest. The levels of another protein involved in cell cycle progression, p53, a mediator in the DNA damage response of cells, was not significantly altered except at the highest level of metolachlor (1,000 ppb) and after a 72-h exposure. These results suggest that the decrease in cell number after low-level metolachlor exposure is most likely due to an alteration in the cell cycle and not due to cell death in human liver cells.