Vitrified before and after genome editing via electroporation.

BACKGROUND: Cryopreservation of bovine zygotes allows for a flexible schedule of genome editing via electroporation. However, vitrification-induced cell membrane damage may not only affect embryonic development but also genome mutation. OBJECTIVE: To investigate the effects of vitrification of zygotes before and after electroporation treatments on the development and genome mutation of bovine presumptive zygotes. MATERIALS AND METHODS: In vitro-derived bovine zygotes were electroporated with the CRISPR/Cas9 system immediately (Vitrified-EP) or 2 h after incubation (Vitrified-2h-EP) following vitrification and warming, or electroporated before vitrification (EP-vitrified). RESULTS: The development rates of vitrified-warmed zygotes were significantly lower (p < 0.05) than those of control zygotes that were not vitrified. Moreover, no differences were observed in the mutation rates and mutation efficiency of the blastocysts resulting from electroporated zygotes, irrespective of the timing of electroporation treatment. CONCLUSION: Our results suggest that vitrification before and after electroporation treatments does not affect the genome editing of zygotes.

Intranasal sensitization of Japanese cedar pollen by the co-administration of low doses of cholera toxin but not its recombinant B subunit to mice.

We evaluated the effects of cholera toxin (CT) and the B subunit of cholera toxin (CTB) on the intranasal sensitization of Japanese cedar pollen (JCP) in mice. JCP suspended in phosphate-buffered saline was administered into the nostrils of mice in combination with varying doses of CT or recombinant CTB(r-CTB) once a week for 5 weeks. Antibody responses specific to sugi basic protein (SBP) were monitored by ELISA for seven weeks. The sensitization of JCP alone did not induce IgG1, IgG2b, IgG2a, IgE or IgA. In contrast, sensitization of JCP in combination with CT (JCP/CT) elicited the prominent production of SBP-specific IgG1 and low levels of IgG2b and IgG2a on Day 49. IgE production was detected only in the serum of mice which were treated with JCP/CT, and not under any other protocol. Using spleen cells from these mice, cytokine production was examined by ELISA in culture supernatants after they had been stimulated in vitro with major cedar pollen allergens, Cry j 1, Cry j 2 or SBP. Notable responses were an increase of IFN-gamma as well as IL-4 in JCP/CT-sensitized cells stimulated with Cry j 2, but not in those stimulated with Cry j 1. No significant differences were detected in IL-5 production among the experimental groups. Histopathological examination, however, showed that eosinophil infiltration was evident in the nasal mucosa of the JCP/CT-sensitized mice following challenge with JCP/CT, but weak with BSA/CT or CT alone. Thus, the immunological and histological analyses indicated that the co-administration of a low dose of CT in combination with JCP allows the induction of pollen-allergic states in mice.

Intraosseous epithelioid malignant peripheral nerve sheath tumor of the phalanx. Case report.

We report the first case of intraosseous epithelioid malignant peripheral nerve sheath tumor (MPNST) occurring in the phalanx. The patient was a 50-year-old Japanese man with an intramedullary lytic lesion of the proximal phalanx. Microscopically, the tumor was composed of epithelioid cells or polygonal cells, forming large cell nests with central necrosis. Most tumor cells were diffusely and strongly immunopositive for S-100 protein and vimentin, and negative for cytokeratin, epithelial membrane antigen, carcinoembryonic antigen, alpha-smooth muscle actin, and HMB-45. Laminin-positive material was discontinuously demonstrated between the individual tumor cells. Electron microscopy showed prominent external lamina. Our case indicated that laminin is useful for differentiating epithelioid MPNST from metastatic carcinoma and malignant melanoma.

Inhibition of M-tropic HIV-1 infection by the fd phage-gene 3 protein with MIP-1alpha-binding activity.

CCR5 is a chemokine receptor with seven transmembrane-domains. It is expressed on T cells and macrophages and functions as the principal co-receptor for macrophage (M)-tropic strains of HIV-1. The anti-CCR5 monoclonal antibody (mAb) 2D7 inhibits the binding and chemotaxis of the three natural beta-chemokine ligands of CCR5, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES, to CCR5(+) cells. The mAb also efficiently blocks the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. In this study, we attempted to determine the peptide motif recognized with the 2D7 mAb. We isolated phage clones by panning a phage display library using 2D7 and identified three peptide motifs. One of these phage clones (M23) showed a marked inhibitory activity on HIV-1 infection. The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the g3p of the M23 phage clone (M23-g3p). The M23-g3p was purified by fast-performance liquid chromatography (FPLC). We show here that (1) M23-g3p was specifically recognized with anti-CCR5 mAb; (2) M23-g3p showed inhibitory activity on the infectivity of M-tropic but not T-tropic HIV-1 strains; (3) M23-g3p bound to MIP-1alpha, MIP-1beta, and RANTES but not MCP-1. These results suggested that the M23-g3p might mimic the CCR5-binding domain shared by beta-chemokines, MIP-1alpha, MIP-1beta, and RANTES as well as the HIV-1 infection.

T cell proliferation-augmenting activities of the gene 3 protein derived from a phage library clone with CD80-binding activity.

We have isolated a phage clone, F2, by panning a phage library with a CTLA4-conformation recognizing mAb (anti-CTLA4 mAb). The unique sequence of 15 amino acids with an internal disulfide bond was inserted in the gene 3 proteins of F2 phage clone (F2-g3p). We show here that 1) F2-g3p was recognized with anti-CTLA4 mAb but not with anti-CD28 mAb, and 2) F2-g3p bound to CD80 but not to CD86. The surface plasmon resonance analysis showed that F2-g3p strongly bound CD80. F2-g3p inhibited the binding of CTLA4 to CD80 but not to CD86. In contrast, F2-g3p weakly inhibited the binding of CD28 with CD80. When hen egg lysozyme (HEL)-primed lymph node cells were stimulated with HEL in the presence of F2-g3p in vitro, cell proliferation was highly potentiated. In the absence of antigenic stimulation, F2-g3p induced no T cell proliferation, indicating the costimulatory nature of F2-g3p. The T cell-augmenting activity of the F2 clone was eliminated when the F2 clone was preincubated with CD80-Ig before the addition to the cultures, indicating the involvement of CD80-binding in the F2-g3p-mediated immunopotentiation. Thus, the F2 motif conferred CD80-binding activity and an immunoregulatory function to the g3p.

Peptide mimics of the CTLA4-binding domain stimulate T-cell proliferation.

Phage library clones selected by a conformational epitope-recognizing and inhibitory monoclonal antibody may display moieties that mimic a receptor/ligand-like three-dimensional structure. This pseudoreceptor/ligand should be able to bind to natural ligand/receptor molecules. We tested this idea using anti-T cell costimulatory molecule antibodies and successfully isolated phage clones with costimulatory effects on T-cell proliferation. This strategy facilitates the designing of regulatory peptide molecules in the absence of precise information about the structure-function relationships in receptor/ligand interactions.