Four immunoglobulin isotypes and IgD splice variants in urodele amphibians.

Until recently, different families of urodele amphibians were thought to express distinct subsets of immunoglobulin (Ig) isotypes. In this study, we explored cDNAs encoding Ig heavy-chains (H-chains) in three species of urodele amphibians. We found that Cynops pyrrhogaster, Pleurodeles waltl, and Ambystoma mexicanum each carry genes encoding four Ig H-chain isotypes, including IgM, IgY, IgD, and IgX, similar to those found in anuran amphibians. We also found that urodele IgDs have a long constant region similar to those found in anuran, reptiles, and bony fishes. We also found several putative IgD splice variants. Our findings indicated that P. waltl IgP is not a novel isotype but an IgD splice variant. Altogether, our findings indicate that IgD splice variants may be universally expressed among amphibian species.

Cepharanthine induces apoptosis through the mitochondria/caspase pathway in murine dendritic cells.

CONTEXT: Cepharanthine (CEP) is a biscoclaurine amphipathic alkaloid isolated from the plant Stephania cepharantha Hayata. Although the effects of CEP on several types of cells have been investigated, those on dendritic cells (DCs) are poorly understood. OBJECTIVE: To investigate the effect of CEP on the induction of apoptosis in murine DCs. MATERIALS AND METHODS: The induction of Annexin V/propidium iodide-positive cells and permeability of mitochondrial membrane potential were evaluated in DCs treated with CEP. Cell-associated caspase activity and DNA fragmentation were analyzed by Dual Sensor: MitoCasp™ and agarose gel electrophoresis, respectively. RESULTS: The number of dead cells was increased by CEP treatment at concentrations more than 10 µg/ml. Flow cytometric analysis revealed that the cell death was found to be apoptosis, CEP treatment reduced mitochondrial membrane potential and upregulated the level of cleaved caspases, including caspase-9 and caspase-3/7, in a dose-dependent fashion. Furthermore, DNA fragmentation was observed in CEP-treated DCs. CONCLUSION: CEP is capable of inducing apoptosis and may be a potential agent against DC-mediated and allergic diseases.

Inhibitory effect of cepharanthine on dendritic cell activation and function.

Dendritic cells (DCs) are specialized antigen presenting cells that connect innate and adaptive immunity. DCs are considered as a major target for controlling excessive immune responses. In this study, the effect of cepharanthine (CEP), a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata, on murine DCs was examined in vitro. CEP inhibited antigen uptake by DCs at a concentration between 1 and 5 µg/ml. Although CEP did not inhibit the expression of costimulatory molecules and major histocompatibility complex (MHC) class I in DCs, the compound inhibited lipopolysaccharide (LPS)-induced DC maturation determined by the expression of costimulatory molecules and MHC class I. In addition, CEP could reduce the production of interleukin-6 and tumor necrosis factor-α in LPS-stimulated DCs. DCs treated with CEP were found to be a poor stimulator of allogeneic T cell proliferation and interferon-γ production from the cells. These results suggest that CEP may have great potential as an immunoregulatory agent against various autoimmune diseases and allergy.

The induction of innate and adaptive immunity by biodegradable poly(γ-glutamic acid) nanoparticles via a TLR4 and MyD88 signaling pathway.

The induction of adaptive immunity through the activation of innate immunity is indispensable for vaccine development. Although strategies for particulate antigen delivery are widely investigated, their immunological mechanisms are unclear. We describe in this study that biodegradable nanoparticles (NPs) elaborated with poly(γ-glutamic acid) (γ-PGA) are able to induce potent innate and adaptive immune responses through Toll-like receptor 4 (TLR4) and MyD88 signaling pathways. The production of inflammatory cytokines from macrophages and the maturation of dendritic cells were impaired in MyD88-knockout and TLR4-deficient mice compared with their wild-types, when the cells were stimulated with γ-PGA NPs. The immunization of these mice with antigen-carrying γ-PGA NPs also resulted in diminished induction of antigen-specific cellular immune responses. These results suggest that γ-PGA NPs have not only an antigen-carrying capacity but also a potent adjuvant function of eliciting adaptive immune responses to the carrying antigen through recognition of the first-line host-sensor system.

Ig L-chain shuffling for affinity maturation of phage library-derived human anti-human MCP-1 antibody blocking its chemotactic activity.

Monocyte chemotactic protein-1 (MCP-1, CC-chemokine ligand 2; CCL2) is involved in the development of various forms of chronic inflammations. Employing the naive human single-chain Fv displaying phage library, we established seven MCP-1-specific scFvs. The MC8 and MC32 clones exhibited blocking activity for the MCP-1-induced chemotaxis of THP-1 cells, in spite of their monovalency. The analysis of V gene usage showed that all clones bore the identical Vh1 gene, IGHV1-24*01, with variable DJ joining sequences, while their Vl usage was relatively varied, suggesting the preferential contribution of the Vh gene. Based on these findings, to minimize the deteriorative influences on the MCP-1 specificity of MC32, we aimed to achieve the affinity maturation of MC32 using MC32 L-chain shuffling library and select MC32 variants. Most MC32 variants increased their affinity by reducing the k(off) value with no influence of the antigen specificity. MC32 variants #22 or #56 showed approximately 15-fold higher affinity than MC32, indicating that the L-chain shuffling library is useful if the Vh is dominantly involved in the determination of the antigen specificity.

Human Fc epsilon RIalpha-specific human single-chain Fv (scFv) antibody with antagonistic activity toward IgE/Fc epsilon RIalpha-binding.

The alpha-chain of Fc epsilon RI (Fc epsilon RIalpha) plays a critical role in the binding of IgE to Fc epsilon RI. A fully human antibody interfering with this interaction may be useful for the prevention of IgE-mediated allergic diseases. Here, we describe the successful isolation of a human single-chain Fv antibody specific to human Fc epsilon RIalpha using human antibody phage display libraries. Using the non-immune phage antibody libraries constructed from peripheral blood lymphocyte cDNA from 20 healthy subjects, we isolated three phage clones (designated as FcR epsilon 27, FcR epsilon 51, and FcR epsilon 70) through two rounds of biopanning selection. The purified soluble scFv, FcR epsilon 51, inhibited the binding of IgE to recombinant Fc epsilon RIalpha, although both FcR epsilon 27 and FcR epsilon 70 showed fine binding specificity to Fc epsilon RIalpha. Since FcR epsilon 51 was determined to be a monomer by HPLC, BIAcore analysis was performed. The dissociation constant of FcR epsilon 51 to Fc epsilon RIalpha was estimated to be 20 nM, i.e., fortyfold lower than that of IgE binding to Fc epsilon RIalpha (K(d) = 0.5 nM). With these characteristics, FcR epsilon 51 exhibited inhibitory activity on the release of histamine from passively sensitized human peripheral blood mononuclear cells.

Regional specification of the head and trunk-tail organizers of a urodele (Cynops pyrrhogaster) embryo is patterned during gastrulation.

The dorsal marginal zone (DMZ) of an amphibian early gastrula is thought to consist of at least two distinct domains: the future head and trunk-tail organizers. We studied the mechanism by which the organizing activities of the lower half of the DMZ (LDMZ) of the urodelean (Cynops pyrrhogaster) embryo are changed. The uninvoluted LDMZ induces the notochord and then organizes the trunk-tail structures, whereas after cultivation in vitro or suramin treatment, the same LDMZ loses the notochord-inducing ability and organizes the head structures. A cell-lineage experiment indicated that the change in the organizing activity of the LDMZ was reflected in the transformation of the inductive ability: from notochord-inducing to neural-inducing activity. Using RT-PCR, we showed that the LDMZ expressed gsc, lim-1, chordin, and noggin, but not the mesoderm marker bra. In the sandwich assay, the LDMZ induced bra expression in the animal cap ectoderm, but the inductive activity was inhibited by cultivation or suramin treatment. The present study indicates that the change in the organizing activity of the LDMZ from trunk-tail to head is coupled with the loss of notochord-inducing activity. Based on these results, we suggest that this change is essential for the specification of the head and trunk-tail organizers during gastrulation.