Syntaxin-4 is essential for IgE secretion by plasma cells

The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.     

B1 Cell IgE Impedes Mast Cell-Mediated Enhancement of Parasite Expulsion through B2 IgE Blockade

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Construction of normal human IgE phage antibody library and the screening of the anti—trichosanthin IgE

Allergen specific IgE response is the major cause of immediate hypersensitivity.However the number of IgEproducing B cells and the amount of IgE,especially the specific IgE,are so low,it greatly impedes the study of the allergic-specifc antibody responses.Here we report the construction of a normal human IgE combinatorial library.The repertoire of IgE VH genes and of κ genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR,and were then constructed to form the phage surface display human Fab(IgEVH) library.A plant protein allergen,trichosanthin(TCS),was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library.Human IgE(Fab) to TCS were detected.     

Renal peroxidative changes mediated by oxalate: the protective role of fucoidan

Oxalate, one of the major constituents of renal stones is known to induce free radicals which damage the renal membrane. Damaged epithelia might act as nidi for stone formation aggravating calcium oxalate precipitation during hyperoxaluria. In the present study, the beneficial effects of fucoidan on oxalate-induced free radical injury were investigated. Male Wistar rats were divided into four groups. Hyperoxaluria was induced in two groups by administration of 0.75% ethylene glycol in drinking water for 28 days and one of them was treated with fucoidan from Fucus vesiculosus at a dose of 5 mg/kg b.wt subcutaneously commencing from the 8th day of induction. A control and drug control (fucoidan alone) was also included in the study. The extent of renal injury in hyperoxaluria was evident from the increased activities of alkaline phosphatase, gamma-glutamyl transferase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase in urine. There was a positive correlation between plasma malondialdehyde levels and renal membrane damage indicating a striking relation between free radical formation and cellular injury. Increased protein carbonyl and decreased thiols further exemplified the oxidative milieu prevailing during hyperoxaluria. Decreased renal membrane ATPases accentuated the renal membrane damage induced by oxalate. Renal microscopic analysis showed abnormal findings in histology as an evidence of oxalate damage. The above biochemical and histopathological discrepancies were abrogated with fucoidan administration, indicating its protective role in oxalate mediated peroxidative injury.     

高效构建卵清白蛋白scFv噬菌体文库及其筛选

目的:建立一种高效噬菌体文库构建方法,获得抗鸡卵清蛋白(ovalbumin,OVA)的单链抗体(scFv)噬菌体展示文库,筛选鉴定获得OVA单链抗体。方法:用OVA蛋白免疫Balb/C小鼠,选取血清抗体效价高的小鼠提取脾脏RNA,利用RT-PCR方法扩增获得小鼠重链和小鼠轻链基因。通过无缝连接酶一步将小鼠重链基因、轻链基因和linker DNA连接起来,插入噬菌体表达载体中,构建OVA scFv噬菌体展示文库。测定文库容量,对文库进行富集筛选,ELISA鉴定阳性克隆,测序后构建真核表达载体,转入Expi-CHO悬浮细胞进行真核表达,利用Western blot进行鉴定。结果:成功获得库容量为1. 2×10~7cfu的OVA scFv噬菌体展示文库,并从中筛选出8个阳性克隆,选取效价最高的2号克隆,在Expi-CHO悬浮细胞中表达获得可溶性抗体。结论:建立了一种高效构建scFv噬菌体文库的方法,筛选获得高结合活性的OVA单链抗体,并成功进行了真核表达,为OVA ELISA检测试剂盒的研制奠定了基础。     

Differentiation of the fucoidan sulfated L-fucose isomers constituents by CE-ESIMS and molecular modeling

Alpha-L-fucose, the monosaccharide component of fucoidan, is found in the polysaccharide mainly as its sulfated form where sulfate groups are in position 2 and/or 4 and/or 3. The correlation between biological activities and structure of fucoidan requires the determination of the sulfation pattern of the fucose residues. Therefore, it is of importance to discriminate between the isobaric sulfated fucose isomers. For this purpose, the three isomers 2-O-, 3-O-, and 4-O-sulfated fucose have been analyzed using electrospray ion trap mass spectrometry and capillary electrophoresis. The results reported herein show that it is possible to differentiate between these three positional isomers of sulfated fucose based on their fragmentation pattern upon MS/MS experiments. 3-O-Sulfated fucose was characterized by the loss of the hydrogenosulfate anion HSO4- as the main fragmentation product, while the two other isomers 2-O-, and 4-O-sulfated fucose exhibited cross-ring fragmentation yielding to distinctive (0,2)X and (0,2)A daughter ion, respectively. A computational study of the conformation of the sulfated fucose isomers was carried out providing an understanding of the fragmentation pattern with respect to the position of the sulfate group.     

Investigation of a sulfate transfer during autohydrolysis of a fucoidan from the brown alga Fucus evanescens by tandem ESIMS

A fucoidan from the brown alga Fucus evanescens was effectively depolymerized by autohydrolysis. Negative-ion electrospray ionization mass spectrometry (ESIMS) revealed that the mixture contained sulfated mono- and oligosaccharides with polymerization degree (DP) up to 6, having from 1 to 4 sulfate groups per molecule. The prevalence of oligosaccharides with even DP was observed. It could be explained by the tendency of the 3-linked α-l-fucopyranose residues to hydrolyze faster than 4-linked ones. The intermolecular sulfate transfer during autohydrolysis was detected by ESIMS, when equimolar quantities of d-Rib and d-Glc were added as acceptors. The products were singly-sulfated and hexose was about four times more effective as an acceptor, than pentose. It was impossible to record MS/MS spectra of the sulfate transfer products, since intensities of their ions were too low.     

Degradation of Laminaria japonica fucoidan by hydrogen peroxide and antioxidant activities of the degradation products of different molecular weights

Conditions of fucoidan degradation by hydrogen peroxide and the antioxidant activities of fucoidans with different molecular weights (Mws) were studied. Fucoidan degradation was monitored by gel permeation chromatography (GPC). Results showed that higher reaction temperature, hydrogen peroxide concentration and reaction time were the main factors that determined the decrease in Mw. There were no significant chemical changes in the backbones of the fucoidans treated with hydrogen peroxide, and the sulphate group contents barely change as compared to the raw polysaccharide. The antioxidant activities of seven fucoidans with different Mws (1.0 kDa, 3.8 kDa, 8.3 kDa, 13.2 kDa, 35.5 kDa, 64.3 kDa, 144.5 kDa) were assessed. The results showed that the relationship between Mws of fucoidans and their antioxidant activities is not simply linear. The samples with Mws of 3.8 kDa, 1.0 kDa and >8.3 kDa have better hydroxyl radical scavenging activity, reducing power and superoxide anion scavenging activity, respectively.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

The potential use of Toll-like receptor agonists to restore the dysfunctional immunity induced by hepatitis C virus

Hepatitis C virus (HCV) infection is a major public health concern with approximately 3% of the world’s population is infected, posing social, economical and health burden. Less than 20% of the infected individuals clear the virus during the acute infection, while the rest develop chronic infection. The treatment of choice for HCV infection is pegylated interferon-α (IFN-α) in combination with ribavarin. Despite the cost and side effects of this treatment regimen, many patients fail this therapy and develop persistent HCV infection, leading to cirrhosis and hepatocellular carcinoma. Although the mechanisms underlying the failure to resolve HCV infection are poorly understood, the incapability of patients to develop effective anti-HCV immunity is a potential cause. We hypothesize that the dysfunctional anti-HCV immunity is due to the emergence of immunosuppressive cells coinciding with a decrease in the stimulatory dendritic cells (DCs) and natural killer (NK) cells. We further hypothesize that applying agents that can correct the imbalance between the immunosuppressive cells and stimulatory cells can results in resolution of chronic HCV. In this review article, we will discuss potential approaches, focusing on the use of Toll-like receptor agonists, to block the suppressive effects of the regulatory cells and restore the stimulatory effects of DCs and NK cells.     

Synergistic augmentation of inflammatory cytokine productions from murine mast cells by monomeric IgE and toll-like receptor ligands

Simultaneous activation of murine mast cells by monomeric IgE and toll-like receptor (TLR) ligands was examined. Inflammatory cytokine production elicited by the binding of IgE in the absence of antigen, was further enhanced by the addition of lipopolysaccharide (LPS) or peptidoglycan (PGN). Enhancement by LPS or PGN on cytokine production was mediated by TLR4 and TLR2, respectively, since TLR4- and TLR2-deficient mast cells did not show synergistic activation by monomeric IgE and LPS/PGN. Synergistic activation of mast cells was obtained via phosphorylation of several mitogen-activated protein kinases (MAPK). Furthermore, MAPK inhibitors, significantly attenuated the augmentation of inflammatory cytokine production by monomeric IgE and LPS or PGN. Altogether, these results suggest that simultaneous TLR activation of mast cells with IgE molecules, particularly highly cytokinergic (HC) IgE, might contribute to the exacerbation of allergic diseases associated with infection even in the absence of a specific antigen.     

Sanguinarine suppresses IgE induced inflammatory responses through inhibition of type II PtdIns 4-kinase(s)

The effects of sanguinarine on IgE mediated early signaling mechanisms leading to inflammatory mediators release were investigated. Pretreatment of RBL 2H3 cells with sanguinarine inhibited IgE induced activation of type II PtdIns 4-kinase activity. Concomitant with type II PtdIns 4-kinase inhibition, sanguinarine also inhibited IgE induced degranulation and β hexosaminidase release in RBL 2H3 cells. In vitro assays showed sanguinarine inhibited type II PtdIns 4-kinase activity in a dose dependent fashion with no effect on PtdIns 3-kinase activity. Fluorescence spectroscopic studies suggested that sanguinarine binds to type II PtdIns 4-kinases α and β isoforms with a K_d of 2.4 and 1.8 μM, respectively. Kinetic studies showed that sanguinarine competes with PtdIns binding site of type II PtdIns 4-kinase β. These results suggest that the anti-inflammatory effects of sanguinarine on PtdIns 3-kinase signaling pathway are more likely an indirect effect and emphasize the importance of the cross talk between type II PtdIns 4-kinases and PtdIns 3-kinases.     

Structural analysis of a fucoidan from the brown alga Fucus evanescens by MALDI-TOF and tandem ESI mass spectrometry

A fucoidan, a heterogeneous sulfated polysaccharide from the brown alga Fucus evanescens, was depolymerized under solvolytic conditions, and its ethanol-extracted low-molecular-weight fraction was analyzed by MALDI-TOFMS and ESIMS/MS. It was found that the mixture contained unsulfated oligosaccharides including some monosulfated components, which were shown to consist of mainly (1→3)-linked 2-O-sulfonated fucose residues (from 1 to 4). Minor components of the mixture were shown to contain 2-O- and 4-O-sulfonated xylose and galactose residues. Among them, mixed monosulfonated fucooligosaccharides were detected and characterized: Xyl-(1→4)-Fuc, Gal-(1→4)-Fuc, Gal-(1→4)-Gal-(1→4)-Fuc, Gal-(1→4)-Gal. Fucose, galactose, and xylose residues were shown to be mainly 2-O-sulfonated with traces of 4-O-sulfonation. Glucuronic acid was also found as a part of non-sulfated fucooligosaccharides: Fuc-(1→3)-GlcA, Fuc-(1→4)-Fuc-(1→3)-GlcA, Fuc-(1→3)-Fuc-(1→4)-Fuc-(1→3)-GlcA.     

Permeability increase in black lipid membrane induced by compound 4880

When compound $\text{48}\text{80}$, a potent histamine liberator, was added in the aqueous phase facing the black lipid membrane, the conductivity of the membrane was remarkably increased. Although valinomycin displayed a distinct selectivity for K⁺ movement, such selection for ionic permeability was not observed in the case of compound $\text{48}\text{80}$.     

IgE production after four routes of injections of Japanese cedar pollen allergen without adjuvant: crucial role of resident cells at intraperitoneal or intranasal injection site in the production of specific IgE toward the allergen

The production of specific IgE antibodies directed toward cedar pollen correlates well with the onset of allergic rhinitis; but the mechanisms of allergen recognition as nonself and Ig class switch to IgE by the immune system are still not fully understood. In the present study, we injected cedar pollen into mice through 4 different routes (intranasal (i.n.), intraperitoneal (i.p.), intravenous (i.v.), and subcutaneous (s.c.)) without adjuvant 1 to 3 times, and determined time-dependent changes in the total and specific serum IgE levels compared with those in the serum levels of other isotype Igs. After an i.p. or i.n. injection of allergen into the mice, they produced a 1.5-to 1.7-fold increase in total IgE, but none in IgG, IgM, or IgA antibodies in their serum, whereas an i.v. or s.c. injection of allergen was inactive as an inducer of total IgE antibodies. Upon a 2nd (s.c.) injection of the allergen into the i.p. or i.n. sensitized mice, a large amount of allergen-specific IgE antibodies was found in the serum. In the case of i.v. or s.c. sensitized mice, however, they produced total, but not specific, IgE antibodies; and a 3rd (s.c.) injection of the allergen resulted in a large amount of specific IgE antibodies in the serum. These results imply that resident cells at the i.p. or i.n. injection site may play a crucial role in the efficient production of total and specific IgE antibodies toward the allergen.