Naoto Yoshino

Co-administration of cholera toxin and apple polyphenol extract as a novel and safe mucosal adjuvant strategy

Although native cholera toxin (CT) is an extremely effective adjuvant, its toxicity prevents its use in humans. We report here that apple polyphenol extract (APE), obtained from unripe apples, reduces CT-induced morphological changes and cAMP accumulation. Based upon this finding, we have attempted to design a novel, effective and safe mucosal vaccine by using CT with several dosages of APE as nasal adjuvants. Mice nasally immunized with OVA plus CT and an optimal dosage of APE showed significantly reduced levels of inflammatory responses as well as total and OVA-specific IgE antibodies when compared with mice given without APE. However, levels of both mucosal and systemic OVA-specific antibody responses were maintained. Further, APE significantly down-regulated accumulation of CT in the olfactory nerves and epithelium. In summary, an optimal dosage of APE would take full advantage of mucosal adjuvanticity of native CT without any toxicity for application in humans.

Polymyxins as Novel and Safe Mucosal Adjuvants to Induce Humoral Immune Responses in Mice

There is currently an urgent need to develop safe and effective adjuvants for enhancing vaccine-induced antigen-specific immune responses. We demonstrate here that intranasal immunization with clinically used polypeptide antibiotics, polymyxin B (PMB) and colistin (CL), along with ovalbumin (OVA), increases OVA-specific humoral immune responses in a dose-dependently manner at both mucosal and systemic compartments. Enhanced immunity by boosting was found to persist during 8 months of observation. Moreover, mice intranasally immunized with OVA plus various doses of PMB or CL showed neither inflammatory responses in the nasal cavity and olfactory bulbs nor renal damages, compared to those given OVA alone. These data suggest that polymyxins may serve as novel and safe mucosal adjuvants to induce humoral immune responses. The polymyxin adjuvanticity was found to be independent of endotoxins liberated by its bactericidal activity, as indicated by similar enhancing effects of PMB in lipopolysaccharide (LPS)-hyporesponsive and LPS-susceptible mice. However, despite the presence of preexisting anti-PMB antibodies, we observed no reduction in the adjuvant function of polymyxins when they were given intranasally. Furthermore, the titers of OVA-specific Abs in mice intranasally immunized with OVA plus PMB or CL were significantly higher than those in mice administered with polymyxin analogues, such as polymyxin B nonapeptide and colistin methanesulfonate. The levels of released β-hexosaminidase and histamine in mast cell culture supernatants stimulated by PMB or CL were also significantly higher than those stimulated by their analogues. These results suggest that both the hydrophobic carbon chain and hydrophilic cationic cyclic peptide contribute to the mucosal adjuvanticity of PMB and CL.

Analysis of the beta-propiolactone sensitivity and optimization of inactivation methods for human influenza H3N2 virus

Beta-propiolactone (BPL) is used as an inactivating reagent for influenza virus in a number of countries. However, the treatment of viruses with BPL occasionally results in a decrease in the hemagglutinin (HA) titer, which complicates vaccine development. In the present study, we examined the biological and biochemical characteristics of human H1N1 and H3N2 viruses treated with BPL, and developed an inactivation method for BPL-sensitive viruses. A significant decrease in HA titer was detected in the H3N2 viruses examined. The decrease in the pH of the virus fluid was not associated with the decreased HA titer, indicating that the decrease in HA titer for the H3N2 virus is the result of the direct effect of BPL. Excessive modification of M1 by BPL and loss of virion diameter were observed in 0.1% BPL-treated H3N2 virus. Taken together, these results suggest that the BPL sensitivity of H3N2 virus results from disruption of the virion. By contrast, the H3N2 virus was successfully inactivated by 0.02% BPL without a significant decrease in the HA titer or disruption of virion structure. Furthermore, we found that the 0.02% BPL in the virion preparation was hydrolyzed successfully by incubation at 37°C for 7h. Thus, mild treatment with a low concentration of BPL enabled us to inactivate the H3N2 virus.

Mast cells partially contribute to mucosal adjuvanticity of surfactin in mice: Mast cells contribute to surfactin adjuvanticity

Surfactin (SF) is a cyclic lipopeptide that has potent mucosal adjuvant properties. However, immunological mechanisms of SF adjuvant action have not yet been elucidated. As some cyclic lipopeptides, such as polymyxin, can stimulate histamine release from mast cells, we hypothesized that mast cell activation is critical for SF adjuvanticity.

Critical micelle concentration and particle size determine adjuvanticity of cyclic lipopeptides

Cyclic lipopeptides such as surfactin and polymyxin have potent mucosal adjuvant properties. Cyclic lipopeptides are tensioactive compounds, but the relationship between adjuvanticity and surface activity is unknown. Here, we show that the critical micelle concentration (cmc) of surfactant and particle size of the surfactant‐protein complex are important determinants of cyclic lipopeptide adjuvanticity. We found that the diameter of cyclic lipopeptide‐ovalbumin (OVA) complex particles was significantly larger than that in the solutions of OVA alone at cyclic lipopeptide concentrations above the cmc. OVA‐specific antibody titres in mice immunized intranasally with OVA and a cyclic lipopeptide at concentrations above its cmc were significantly higher than those in mice immunized with OVA plus the same dose of the cyclic lipopeptide but administered with formulations in which cyclic lipopeptide concentration was below the cmc. Thus, the concentration of the cyclic lipopeptide in the formulation at immunization, but not its overall dose, was critical for its adjuvanticity. Furthermore, two types of aggregates, the cyclic lipopeptide simplex micelles and the cyclic lipopeptide‐OVA complex micelles, were found in formulations with SF concentrations above its cmc. Degranulation of mast cells exposed to SF simplex micelles was more pronounced when SF concentration was above the cmc. In conclusion, our study showed that surface activity properties, such as the cmc and the size of surfactant‐protein complex, contribute to the adjuvanticity of cyclic lipopeptides. Our study proposes a novel idea that cmc is a key parameter for tensioactive adjuvants.

Optimization of trypsins for influenza A/H1N1 virus replication in MDCK SI-6 cells, a novel MDCK cell line

A cell-based vaccine production method for influenza virus may be an effective and more rapid alternative to egg-based systems. For high-yield virus production, the effect of bovine, porcine, fungal, and recombinant trypsins on influenza A/H1N1 virus replication in MDCK SI-6 cells (SI-6 cells), a novel MDCK cell line developed by our research group, was examined. SI-6 cells infected with influenza A/H1N1 virus were incubated in the presence of four trypsin types at various concentrations, and virus yields in the culture medium were evaluated by a hemagglutination (HA) assay. Virus growth was most efficient in the presence of bovine and porcine trypsins. An analysis of the optimized concentration and definitive HA titer of each trypsin by Gaussian distribution revealed that comparable high virus yields (166.1 and 164.2 HAU/50μl) were obtained at the optimized concentrations of bovine (0.4μg/ml) and porcine (2.1μg/ml) trypsins, respectively, the yields of which were significantly higher than that of fungal and recombinant trypsins. We conclude that bovine and porcine trypsins are suitable for influenza A/H1N1 virus replication in SI-6 cells. This result complements our previous study and suggests the possible application of SI-6 cells to the development of cell-based influenza vaccines.

Establishment of a cell model of X-linked sideroblastic anemia using genome editing

ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patients bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.

Inhibition of Aurora Kinase A Synergistically Enhances Cytotoxicity in Ovarian Clear Cell Carcinoma Cell Lines Induced by Cisplatin: A Potential Treatment Strategy

Objective This study aims to clarify the incidence of Aurora kinase A (Aurora-A) protein expression and its correlation with clinical parameters in ovarian clear cell carcinoma (OCCC) tumor tissues. In addition, we assessed the efficacy of ENMD-2076, a novel selective Aurora-A inhibitor, in combination with chemotherapeutic agents for the treatment of OCCC. Methods/Materials Aurora-A protein expression was determined by immunohistochemical staining of OCCC specimens from 56 patients to evaluate its correlation with clinical outcomes in OCCC. In the in vitro study, 6 OCCC cell lines were exposed to ENMD-2076 in combination with cisplatin, SN38, doxorubicin, or paclitaxel, and cell proliferation, cell cycle distribution, and apoptosis were assessed. Results The 5-year survival rates of International Federation of Gynecology and Obstetrics stages IC3 to IV patients with intermediate or strong Aurora-A expression were significantly lower than those of patients with negative or weak Aurora-A expression. Increased Aurora-A expression was associated with significantly worse overall survival of International Federation of Gynecology and Obstetrics stages IC3 to IV patients (21% vs 77%). Multivariate analysis revealed that Aurora-A expression was an independent prognostic factor for stages IC3 to IV OCCC patients. Furthermore, synergistic effects were observed with ENMD-2076 in combination with cisplatin or SN-38 in 4 of the 6 tested cell lines. ENMD-2076 dramatically enhanced apoptosis and cell cycle arrest at the G2/M phase induced by cisplatin. Conclusions Aurora-A is a promising biomarker that is predictive of patient outcomes and a potential target for OCCC. The results suggested that chemotherapy, including ENMD-2076 in combination with cisplatin, is a potential treatment modality for patients with OCCC.