Bioleaders (South Korea)

Abstract Introduction : The poly-gamma-glutamic acid (γ-PGA) is a safe and edible polypeptide that is naturally synthesized by Bacillus subtilis 1 and has long been taken up as an ingredient of fermented soybean foods. The γ-PGA as a signaling regulator of Toll-like receptor 4 (TLR4), has been shown to enhance natural killer (NK) cell activity and inhibit tumors. Although CIN-1 typically represents transient infection, about 10% of CIN-1 is found to progress to CIN-3, and HPV infection persists and leads to high grade cervical dysplasia in some women with CIN-1. The aim of this study was to investigate the efficacy and safety of γ-PGA and impact γ-PGA on the immune system including PD-1 / PD-L1 in patients with CIN 1. Material & Methods : We conducted a multi-center, randomized, double blind, placebo control, parallel designed, phase 2b study. Participants were randomly assigned to one of two groups: half received oral solution containing 1500 mg of γ-PGA; half received a placebo. Each participants took one dose every 24 hours for 4 weeks. Colposcopy with biopsy was assessed at the time of screening and 12 weeks. Blood samples were drawn at the initial treatment and at the 4th, 8th, 12th weeks after treatment. The primary endpoint of the study was histologic regression rate between screening and 12th weeks. The secondary endpoint were cytologic response rate, HPV clearance, change of immune responses and PD-1/PD-L1 expression. Result From April 2013 to December 2015, 254 patients were screened and a total of 200 patients participated in the study. Of these, Of 101 women in γ-PGA group, 42 (42.4%) women experienced histologic regression, whereas 26 (27.1%) in the control group experienced histologic regression, showing statistically significant differences (p=0.018). In the γ- PGA group, HPV clearance was found in 37 (43.5%) of the 85 high-risk HPV infected patients, showing a meaningful difference compared to the control group showing HPV clearance of 20 (26.7%) out of 75 (p=0.026). However, there was no significant difference between the two groups in terms of cytologic regression rate and type specific clearance of HPV. The immunologic factors including NK cell activity, CD8 cell count and PD-1/PD-L1 expression, which are thought to be related to the therapeutic mechanism of γ- PGA, showed no significant difference between the two groups. Conclusion : The γ-PGA enhance histologic regression in CIN-1 patients and showed the possibility of non-invasive medical treatment. In addition, γ-PGA has shown its potential as a treatment for high-grade CIN or cancer in the future as it has been shown to be effective against high-risk HPV infection. Citation Format: Hyun-Woong Cho, Yung-Taek Ouh, Jin Hwa Hong, Jae Kwan Lee, Jae-Won Jeon, Moon-Hee Sung. Clinical and immunologic results of a multi-center, randomized, double blind, phase II trial using poly-gamma-glutamic acid (γ-PGA) for women with cervical intraepithelial neoplasia 1 (CIN 1) and correlation of PD-1/PD-L1 tumor expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT080.

Photodynamic therapy (PDT) is a treatment option for skin cancer and premalignant skin diseases and exhibits rejuvenation effects, including reducing fine wrinkles and whitening, on aged skin. In this study, we investigated the mechanism underlying the whitening effects of PDT on melanocytes (MCs) in vitro and in vivo. Exposure of MCs to PDT in vitro reduced their melanin content and tyrosinase activity without, however, affecting cell survival. Interestingly, melanogenesis was also inhibited by exposing MCs to conditioned media of PDT-treated keratinocytes or dermal fibroblasts. This paracrine effect was likely due to a decreased release of melanocyte-stimulating cytokines such as Kit ligand and hepatocyte growth factor from these cells. Furthermore, we observed that PDT reduced mottled hyperpigmentation of photoaged patient skin in vivo, highlighting the clinical importance of skin whitening by PDT.

In 2009, the global outbreak of an influenza pandemic emphasized the need for an effective vaccine adjuvant. In this study, we examined the efficacy of poly-γ-glutamic acid/chitosan (PC) nanogel as an adjuvant for the influenza vaccine. PC nanogel significantly enhanced antigen-specific cross-presentation and cytotoxic T lymphocyte (CTL) activity. Compared with alum, the protective efficacy of the pandemic H1N1 influenza (pH1N1) vaccine was substantially increased by PC nanogel, with increased hemagglutination-inhibition titers, CTL activity, and earlier virus clearance after homologous and heterosubtypic [A/Philippines/2/82 (H3N2)] virus challenges. However, CD8+ T cell-depleted mice displayed no protection against the heterosubtypic virus challenge after immunization with PC nanogel-adjuvanted pH1N1 vaccine. We also observed that using PC nanogel as a vaccine adjuvant had a dose-sparing effect and significantly enhanced the long-lasting protection of the pH1N1 vaccine. Together, these results suggest that PC nanogel is a promising vaccine adjuvant that could broadly prevent influenza virus infection.

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.

An antibacterial protein (about 12 kDa) was isolated from human amniotic fluid through dialysis, ultrafiltration and C18 reversed-phase HPLC steps. Automated Edman degradation showed that the N-terminal sequence of the antibacterial protein was NH2-Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-. The N-terminal sequence of the antibacterial protein was found to be identical to that of β2-microglobulin, a component of MHC class I molecules, which are present on all nucleated cells. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) revealed that the molecular mass of the antibacterial protein was 11,631 Da. This antibacterial protein, β2M, possessed potent antibacterial activity against pathogenic bacteria. Specially, antibacterial activity was observed in potassium buffer, and potassium ion was found to be critical for the antibacterial activity. Interestingly, the antibacterial action of β2M was associated with dissipation of the transmembrane potential, but the protein did not cause damage to the membrane that would result in SYTOX green uptake. In addition, stimulation of WISH amniotic epithelial cells with the bacterial endotoxin lipopolysaccharide (LPS) induced dose-dependent upregulation of β2M mRNA expression. These results suggest that β2M contributes to a self-defense response when amniotic cells are exposed to pathogens.

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA(225G) and NA(315N) substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA(225G) and NA(315N) as potential virulence markers in mammals.

It is present herein an algorithm for thresholding images of bank checks. These images have complex background elements. Some of these patterns make very hard to distinguish between the text and the texture pattern defined by the bank. For the binarizing process, an adaptive global thresholding algorithm is proposed based on ROC curves and it is compared to several well-known algorithms. The images generated by the new algorithm achieved a hit rate of 97% for recognition of the CMC7 code.

We analyzed the in vivo tumor regression activity of high molecular mass poly-gamma-glutamate (gamma-PGA) from Bacillus subtilis sups. chungkookjang. C57BL/6 mice were orally administered 10-, 100-, or 2000-kDa gamma-PGA or beta-glucan (positive control), and antitumor immunity was examined. Our results revealed higher levels of NK cell-mediated cytotoxicity and IFN-gamma secretion in mice treated with higher molecular mass gamma-PGA (2000 kDa) vs those treated with lower molecular mass gamma-PGA (10 or 100 kDa) or beta-glucan. We then examined the effect of oral administration of 10- or 2000-kDa gamma-PGA on protection against B16 tumor challenge in C57BL/6 mice. Mice receiving high molecular mass gamma-PGA (2000 kDa) showed significantly smaller tumor sizes following challenge with the MHC class I-down-regulated tumor cell lines, B16 and TC-1 P3 (A15), but not with TC-1 cells, which have normal MHC class I expression. Lastly, we found that gamma-PGA-induced antitumor effect was decreased by in vivo depletion of NK cells using mAb PK136 or anti-asialo GM1 Ab, and that was completely blocked in NK cell-deficient B6 beige mice or IFN-gamma knockout mice. Taken together, we demonstrated that oral administration of high molecular mass gamma-PGA (2000 kDa) generated significant NK cell-mediated antitumor activity in mice bearing MHC class I-deficient tumors.