Shuhei Hashiguchi

Molecular characterization of a 10‐kDa buckwheat molecule reactive to allergic patients’ IgE

Background:  Using the sera from buckwheat (BW)‐allergic patients, several putative causative molecules were reported. However, few molecules were determined on the molecular structure. We demonstrated in 2000 that the major allergen with 24 kDa (BW24KD) is a legumin‐like storage protein.

Th1/Th2 response profiles to the major allergens Cry j 1 and Cry j 2 of Japanese cedar pollen.

Cry j 1 and Cry j 2 are known to be the major allergens of Japanese cedar pollen. A comparative study was carried out on the immune responses to stimulation with Cry j 1 and Cry j 2 in 24 symptomatic patients and six nonallergic subjects. In T‐cell proliferation assays, mean stimulation indexes (SI) were 10.6 for Cry j 1 and 11.7 for Cry j 2 stimulation, respectively, in the allergic patients. Two of the nonallergic subjects showed strong T‐cell proliferation to both allergens, while the remainder did not. All the allergic subjects (17/17) showed high titers of anti‐Cry j 1 IgE antibody at a mean value of 165 U/ml, whereas only 64% responded to Cry j 2 with low titers at a mean value of 26 U/ml. Nonallergic subjects did not respond with IgE production. Allergic subjects were further examined for their cytokine production profiles. All allergic subjects tested (16/16) produced high levels of interferon‐gamma (IFN‐γ) in response to Cry j 1 with a mean value of 918 pg/ml, while only five subjects showed significant elevation of IFN‐γ production in response to Cry j 2 with a mean value of 679 pg/ml. The remainder produced small amounts of IFN‐γ. Cry j 1 induced higher levels of interleukin (1L)‐10 gene expression than did Cry j 2 stimulation, while both allergens induced IL‐4 expression at a similar level. The IL‐12 p35 gene was constitutively expressed, whereas the IL‐12 p40 gene expression in Cry j 1‐stimulated cells was elevated eightfold over that of nonstimulated cells. Increased expression of the IL‐12 p40 gene was negligible in Cry j 2‐stimulated cells. Thus, Cry j 1 stimulated mixed features of Th1 and Th2‐like responses, while Cry j 2 played a minor role in inducing IgE production and cytokine (IFN‐γ, IL‐10, and IL‐12) production, except for IL‐2 production and strong T‐cell proliferative activity. Therefore, it was concluded that Cry j 1 is the more important allergen, and that T‐cell proliferation assays do not necessarily reflect the level of allergenicity.

Immunodominance of seven regions of a major allergen, Cry j 2, of Japanese cedar pollen for T-cell immunity

The immunodominant regions of the Japanese cedar pollen allergen Cry j 2 for T‐cell immunity were determined with whole peripheral blood lymphocytes (PBL) derived from seven allergic patients and three nonallergic subjects. Cry j 2‐stimulated T‐cell proliferation was inhibited by anti‐HLA‐DR. but not by anti‐HLA‐DQ antibody, indicating that the responding T cells recognized the allergen peptides associated with HLA‐DR molecules. It was found that seven regions of Cry j 2, i.e., regions corresponding to amino acid numbers 1–26, 70–84, 151–167. 187–203, 252–279, 283–314, and 345–362, were immunodominant for T‐cell proliferation. Thus, Cry j 2 bears a limited number of immunodominant regions despite polymorphic features of HLA‐DR in the immune system. This suggests the possibility of molecularly designing Cry j 2 antagonists that could downregulate allergic reactions to Japanese cedar pollen.

Immunological basis of M13 phage vaccine: Regulation under MyD88 and TLR9 signaling

Peptide-displaying bacteriophages induce mimotope-specific antibody responses, suggesting a novel application of phage-display library as bacteriophage vaccine. We examined the antibody response against M13 phage in mice induced by an i.p. administration of M13 phage in phosphate-buffered saline. We showed here that firstly, mice showed strong IgG antibody responses, particularly, in IgG2b, IgG2c, and IgG3 subclasses even in primary responses. Secondly, IgG production in primary response is totally dependent on MyD88 signaling. These responses were almost comparable, but slightly weaker, in TLR2-, TLR4- and TLR7-deficient mice relative to wild-type mice, suggesting that this enhancing effect is not due to plausible LPS contamination. Thirdly, although primary IgG1 response was not detected in wild-type mice, remarkable IgG1 response was induced in TLR9-deficient mice, suggesting that TLR9 pathway functions as regulatory, but not a simple augmenting signaling cascade, and furthermore, the enhanced IgG1 response was not due to adjuvant effect of single-stranded DNA derived from M13 phage. Thus, innate immunity including TLR regulation is crucial for M13 phage vaccine design.

Reversible monomer-oligomer transition in human prion protein

The structure and the dissociation reaction of oligomers PrPoligo from reduced human prion huPrPC (23-231) have been studied by 1H-NMR and tryptophan fluorescence spectroscopy at varying pressure, along with circular dichroism and atomic force microscopy. The 1H-NMR and fluorescence spectral feature of the oligomer is consistent with the notion that the N-terminal residues including all seven Trp residues, are free and mobile, while residues 105~210, comprising the AGAAAAGA motif and S1-Loop-HelixA-Loop-S2-Loop-HelixC, are engaged in intra- and/or inter-molecular interactions. By increasing pressure to 200 MPa, the oligomers tend to dissociate into monomers which may be identified with PrPC*, a rare metastable form of PrPC stabilized at high pressure (Kachel et al. BMC. Struct. Biol. 6, 16). The results strongly suggest that the oligomeric form PrPoligo is in dynamic equilibrium with the monomeric forms via PrPC*, namely huPrPC ⇄ huPrPC* ⇄ huPrPoligo.

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.

Immunoreactivity of Phage Library-derived Human Single-Chain Antibodies to Amyloid Beta Conformers In Vitro

The pathogenesis of Alzheimers disease involves conformational changes of A beta. A series of antibodies recognizing a distinct conformation of A beta (snapshot antibody) is useful for both understanding the mechanism of molecular conversion and identifying diagnostic and therapeutic reagents. As A beta with various conformations can be prepared in vitro under varying physicochemical conditions, snapshot antibodies can be isolated by directly binding to target molecules with antibody-displaying phages. We tested the feasibility of this idea. We show a feature of several A beta-reactive antibodies isolated from our human single-chain Fv antibody-phage library and particularly report the characteristics of an scFv clone, B6, selected from the fibrillar A beta 1-42-coated biopanning. B6 bound to fibrillar A beta 1-42 as well as globulomer A beta 1-42 but not to soluble A beta 1-42 or A beta 1-40. B6 inhibited A beta 1-42 fibril formation with 600 nM IC50 in spite of being the monovalent scFv form. Epitope analysis suggested that the binding site might be located at the beta2 sheet of the C-terminus of A beta 1-42. Although it is believed that N-terminus-recognizing antibodies tend to show the capability to inhibit A beta 1-42 fibrillation, B6 is the first human inhibitory antibody recognizing the C-terminus of A beta 1-42.

Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis.

Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-α), interleukin 12p40 (IL-12), and interferon gamma (IFN-γ) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-α, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC.

A mimotope peptide of Aβ42 fibril-specific antibodies with Aβ42 fibrillation inhibitory activity induces anti-Aβ42 conformer antibody response by a displayed form on an M13 phage in mice.

In Alzheimers disease (AD), amyloid-β (Aβ) peptides accumulate in the brain in different forms, including fibrils and oligomers. Recently, we established three distinct conformation-dependent human single-chain Fv (scFv) antibodies, including B6 scFv, which bound to Aβ42 fibril but not to soluble-form Aβ, inhibiting Aβ42 fibril formation. In this study, we determined the mimotopes of these antibodies and found a common mimotope sequence, B6-C15, using the Ph.D.-C7C phage library. The B6-C15 showed weak homology to the C-terminus of Aβ42 containing GXXXG dimerization motifs. We synthesized the peptide of B6-C15 fused with biotinylated TAT at the N-terminus (TAT-B6-C15) and characterized its biochemical features on an Aβ42-fibrillation reaction in vitro. We demonstrated that, first, TAT-B6-C15 inhibited Aβ42 fibril formation; secondly, TAT-B6-C15 bound to prefibril Aβ42 oligomers but not to monomers, trimers, tetramers, fibrils, or ultrasonicated fragments; thirdly, TAT-B6-C15 inhibited Aβ42-induced cytotoxicity against human SH-SY5Y neuroblastoma cells; and, fourthly, when mice were administered B6-C15-phages dissolved in phosphate-buffered saline, the anti-Aβ42 conformer IgG antibody response was induced. These results suggested that the B6-C15 peptide might provide unique opportunities to analyze the Aβ42 fibrillation pathway and develop a vaccine vehicle for Alzheimers disease.

Direct Evidence of Generation and Accumulation of β-Sheet-rich Prion Protein in Scrapie-infected Neuroblastoma Cells with Human IgG1 Antibody Specific for β-Form Prion Protein

Background: PrPSc is believed to have a β-sheet-rich structure. Results: Established human IgG1 stained β-form PrP in prion-infected cells but had no inhibitory activity against the propagation of PrPres. Conclusion: β-Sheet-rich PrP was generated and accumulated in cells. Significance: β-Form PrP aggregates play roles in cytotoxicity, whereas prion or PrPSc is responsible for prion propagation. We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935–1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrPSc with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.