Beraprost enhances the APC function of B cells by upregulating CD86 expression levels

Lipid mediators are emerging as important regulators of the immune system. Based on our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Owing to the very short half-life of prostacyclin in experimental conditions, we used a more stable analog, beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE(2), 6-keto-PGF(1α), and PGF(2α) failed to alter CD86 expression levels, whereas other prostacyclin analogs were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allogeneic T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.     

A tyrosine-sulfated CCR5-mimetic peptide promotes conformational transitions in the HIV-1 envelope glycoprotein

The HIV-1 envelope glycoprotein is a trimeric complex of heterodimers composed of a surface glycoprotein, gp120, and a transmembrane component, gp41. The association of this complex with CD4 stabilizes the coreceptor-binding site of gp120 and promotes the exposure of the gp41 helical region 1 (HR1). Here, we show that a 15-amino-acid peptide mimetic of the HIV-1 coreceptor CCR5 fused to a dimeric antibody Fc domain (CCR5mim-Ig) bound two gp120 molecules per envelope glycoprotein complex and by itself promoted HR1 exposure. CCR5mim-Ig also stabilized the association of a CD4-mimetic peptide with the envelope glycoprotein. A fusion of the CD4- and CCR5-mimetic peptides, DM1, bound gp120 and neutralized R5, R5X4, and X4 HIV-1 isolates comparably to CD4, and they did so markedly more efficiently than either peptide alone. Our data indicate that the potency of DM1-Ig derives from its avidity for the HIV-1 envelope glycoprotein trimer and from the bidirectional induction of its receptor-mimetic components. DM1 has significant advantages over other inhibitors that target both coreceptor and CD4-binding sites, and it may serve as a lead for a new class of HIV-1 inhibitor peptides.     

Mouse Hepatitis Virus Infection Upregulates Genes Involved in Innate Immune Responses

Neurotropic recombinant strain of Mouse Hepatitis Virus, RSA59, induces meningo-encephalitis, myelitis and demyelination following intracranial inoculation. RSA59 induced neuropathology is partially caused by activation of CNS resident microglia, as demonstrated by changes in cellular morphology and increased expression of a microglia/macrophage specific calcium ion binding factor, Iba1. Affymetrix Microarray analysis for mRNA expression data reveals expression of inflammatory mediators that are known to be released by activated microglia. Microglia-specific cell surface molecules, including CD11b, CD74, CD52 and CD68, are significantly upregulated in contrast to CD4, CD8 and CD19. Protein analysis of spinal cord extracts taken from mice 6 days post-inoculation, the time of peak inflammation, reveals robust expression of IFN-γ, IL-12 and mKC. Data suggest that activated microglia and inflammatory mediators contribute to a local CNS microenvironment that regulates viral replication and IFN-γ production during the acute phase of infection, which in turn can cause phagolysosome maturation and phagocytosis of the myelin sheath, leading to demyelination.     

Establishment and characterization of a lung cancer cell line,SMC-L001, from a lung adenocarcinoma

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.     

Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD⁺. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K _m) of 5.87 μM, a turnover rate (k _cat) of 0.24/sec, and a catalytic efficiency (k _cat/K _m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k _cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k _cat/K _m, 107.75/mM/sec).     

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcϵRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcϵRI signaling in lung mast cells in an HDAC3-dependent manner. FcϵRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.     

TuJ1 (class III beta-tubulin) expression suggests dynamic redistribution of follicular dendritic cells in lymphoid tissue

Follicular dendritic cells (FDCs) play central roles in the B cell survival, proliferation, and differentiation into memory cells. Here, we show that TuJ1 (class III beta-tubulin) is expressed strongly in FDCs of human lymphoid tissue. TuJ1 has been a marker of neurons in the central and peripheral nervous systems from the early stage of neural differentiation. FDCs expressed TuJ1 protein diffusely in both light and dark zones of germinal centers in all human lymphoid tissues. In contrast, CD21 expression was relatively concentrated to the light zone, suggesting that TuJ1 was a marker for FDCs with broader spectrum than CD21. In addition to the germinal center, there were single TuJ1-expressing cells scattered in the mantle zone, blurring the border of the FDC network. In human tonsils, single scattered TuJ1-positive cells were also present in the crypt epithelium, suggesting a dynamic redistribution of FDCs among the antigen-rich epithelium, mantle zone, and germinal center. Such migration of FDCs could reflect a way of direct transport of various antigens carried on their surface to the germinal center, and a basis for the polarity of lymphoid follicles toward the epithelium in mucosa-associated lymphoid tissues. HK cells, cultured FDCs, also expressed TuJ1. The expression of TuJ1 by FDCs suggests that they may share certain biological characteristics of the neural system.     

Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli

The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His-tagged model proteins: gloshedobin, a thrombin-like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine-induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost-prohibitive spermine and EDTA. The residual PEI in the post-extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI-mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.