Development and Validation of an Enzyme-Linked Immunosorbent Assay-Based Protocol for Evaluation of Respiratory Syncytial Virus Vaccines.

Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 µg/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r > 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.

The combination of calreticulin-targeting L-ASNase and anti-PD-L1 antibody modulates the tumor immune microenvironment to synergistically enhance the antitumor efficacy of radiotherapy.

Radiotherapy (RT) triggers immunogenic cell death (ICD). L-ASNase, which catalyzes the conversion of asparagine (Asn), thereby depleting it, is used in the treatment of blood cancers. In previous work, we showed that CRT3LP and CRT4LP, PASylated L-ASNases conjugated to the calreticulin (CRT)-specific monobodies CRT3 and CRT4, increase the efficacy of ICD-inducing chemotherapy. Here, we assessed their efficacy in tumor-bearing mice treated with RT. Methods: Monobody binding was evaluated by in silico molecular docking analysis. The expression and cellular localization of ecto-CRT were assessed by confocal imaging and flow cytometry. The antitumor effect and the roles of CRT3LP and CRT4LP in irradiation (IR)-induced ICD in tumors were analyzed by ELISA, immunohistochemistry, and immune analysis methods. Results: Molecular docking analysis showed that CRT3 and CRT4 monobodies were stably bound to CRT. Exposure to 10 Gy IR decreased the viability of CT-26 and MC-38 tumor cells in a time-dependent manner until 72 h, and increased the expression of the ICD marker ecto-CRT (CRT exposed on the cell surface) and the immune checkpoint marker PD-L1 until 48 h. IR enhanced the cytotoxicity of CRT3LP and CRT4LP in CT-26 and MC-38 tumor cells, and increased reactive oxygen species (ROS) levels. In mice bearing CT-26 and MC-38 subcutaneous tumors treated with 6 Gy IR, Rluc8-conjugated CRT-specific monobodies (CRT3-Rluc8 and CRT4-Rluc8) specifically targeted tumor tissues, and CRT3LP and CRT4LP increased total ROS levels in tumor tissues, thereby enhancing the antitumor efficacy of RT. Tumor tissues from these mice showed increased mature dendritic, CD4+ T, and CD8+ T cells and pro-inflammatory cytokines (IFNγ and TNFα) and decreased regulatory T cells, and the expression of tumor cell proliferation markers (Ki67 and CD31) was downregulated. These data indicate that the combination of IR and CRT-targeting L-ASNases activated and reprogramed the immune system of the tumor microenvironment. Consistent with these data, an immune checkpoint inhibitor (anti-PD-L1 antibody) markedly increased the therapeutic efficacy of combined IR and CRT-targeting L-ASNases. Conclusion: CRT-specific L-ASNases are useful as additive drug candidates in tumors treated with RT, and combination treatment with anti-PD-L1 antibody increases their therapeutic efficacy.

Gamma Irradiation-Inactivated Respiratory Syncytial Virus Vaccine Provides Protection but Exacerbates Pulmonary Inflammation by Switching from Prefusion to Postfusion F Protein.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes lower respiratory diseases among infants and elderly people. Moreover, formalin-inactivated RSV (FI-RSV) vaccine induces serious enhanced respiratory disease (ERD). Radiation has been investigated as an alternative approach for producing inactivated or live-attenuated vaccines, which enhance the antigenicity and heterogeneous protective effects of vaccines compared with conventional formalin inactivation. In this study, we developed an RSV vaccine using gamma irradiation and analyzed its efficacy against RSV vaccine-induced ERD in a mouse model. Although gamma irradiation-inactivated RSV (RI-RSV) carbonylation was lower than FI-RSV carbonylation and RI-RSV showed a significant antibody production and viral clearance, RI-RSV caused more obvious body weight loss, pulmonary eosinophil infiltration, and pulmonary mucus secretion. Further, the conversion of prefusion F (pre-F) to postfusion F (post-F) was significant for both RI-RSV and FI-RSV, while that of RI-RSV was significantly higher than that of FI-RSV. We found that the conversion from pre- to post-F during radiation was caused by radiation-induced reactive oxygen species. Although we could not propose an effective RSV vaccine manufacturing method, we found that ERD was induced by RSV vaccine by various biochemical effects that affect antigen modification during RSV vaccine manufacturing, rather than simply by the combination of formalin and alum. Therefore, these biochemical actions should be considered in future developments of RSV vaccine. IMPORTANCE Radiation inactivation for viral vaccine production has been known to elicit a better immune response than other inactivation methods due to less surface protein damage. However, we found in this study that radiation-inactivated RSV (RI-RSV) vaccine induced a level of immune response similar to that induced by formalin-inactivated RSV (FI-RSV). Although RI-RSV vaccine showed less carbonylation than FI-RSV, it induced more conformational changes from pre-F to post-F due to the gamma radiation-induced reactive oxygen species response, which may be a key factor in RI-RSV-induced ERD. Therefore, ERD induced by RSV vaccine may be due to pre-F to post-F denaturation by random protein modifications caused by external stress. Our findings provide new ideas for inactivated vaccines for RSV and other viruses and confirm the importance of pre-F in RSV vaccines.

Low-dose radiation therapy suppresses viral pneumonia by enhancing broad-spectrum anti-inflammatory responses via transforming growth factor-ß production.

Low-dose radiation therapy (LDRT) can suppress intractable inflammation, such as that in rheumatoid arthritis, and is used for treating more than 10,000 rheumatoid arthritis patients annually in Europe. Several recent clinical trials have reported that LDRT can effectively reduce the severity of coronavirus disease (COVID-19) and other cases of viral pneumonia. However, the therapeutic mechanism of LDRT remains unelucidated. Therefore, in the current study, we aimed to investigate the molecular mechanism underlying immunological alterations in influenza pneumonia after LDRT. Mice were irradiated to the whole lung 1 day post-infection. The changes in levels of inflammatory mediators (cytokines and chemokines) and immune cell populations in the bronchoalveolar lavage (BALF), lungs, and serum were examined. LDRT-treated mice displayed markedly increased survival rates and reduced lung edema and airway and vascular inflammation in the lung; however, the viral titers in the lungs were unaffected. Levels of primary inflammatory cytokines were reduced after LDRT, and transforming growth factor-ß (TGF-ß) levels increased significantly on day 1 following LDRT. Levels of chemokines increased from day 3 following LDRT. Additionally, M2 macrophage polarization or recruitment was increased following LDRT. We found that LDRT-induced TGF-ß reduced the levels of cytokines and polarized M2 cells and blocked immune cell infiltration, including neutrophils, in BALF. LDRT-induced early TGF-ß production was shown to be a key regulator involved in broad-spectrum anti-inflammatory activity in virus-infected lungs. Therefore, LDRT or TGF-ß may be an alternative therapy for viral pneumonia.

Radioprotection of deinococcal exopolysaccharide BRD125 by regenerating hematopoietic stem cells.

There is a substantial need for the development of biomaterials for protecting hematopoietic stem cells and enhancing hematopoiesis after radiation damage. Bacterial exopolysaccharide (EPS) has been shown to be very attractive to researchers as a radioprotectant owing to its high antioxidant, anti-cancer, and limited adverse effects. In the present study, we isolated EPS from a novel strain, Deinococcus radiodurans BRD125, which produces EPS in high abundance, and investigated its applicability as a radioprotective biomaterial. We found that EPS isolated from EPS-rich D. radiodurans BRD125 (DeinoPol-BRD125) had an excellent free-radical scavenging effect and reduced irradiation-induced apoptosis. In addition, bone-marrow and spleen-cell apoptosis in irradiated mice were significantly reduced by DeinoPol-BRD125 administration. DeinoPol-BRD125 enhanced the expression of hematopoiesis-related cytokines such as GM-CSF, G-GSF, M-CSF, and SCF, thereby enhancing hematopoietic stem cells protection and regeneration. Taken together, our findings are the first to report the immunological mechanism of a novel radioprotectant, DeinoPol-BRD125, which might constitute an ideal radioprotective and radiation mitigating agent as a supplement drug during radiotherapy.

Development of Live Attenuated Salmonella Typhimurium Vaccine Strain Using Radiation Mutation Enhancement Technology (R-MET).

Salmonella enterica is a leading cause of food-borne diseases in humans worldwide, resulting in severe morbidity and mortality. They are carried asymptomatically in the intestine or gallbladder of livestock, and are transmitted predominantly from animals to humans via the fecal-oral route. Thus, the best preventive strategy is to preemptively prevent transmission to humans by vaccinating livestock. Live attenuated vaccines have been mostly favored because they elicit both cellular and humoral immunity and provide long-term protective immunity. However, developing these vaccines is a laborious and time-consuming process. Therefore, most live attenuated vaccines have been mainly used for phenotypic screening using the auxotrophic replica plate method, and new types of vaccines have not been sufficiently explored. In this study, we used Radiation-Mutation Enhancement Technology (R-MET) to introduce a wide variety of mutations and attenuate the virulence of Salmonella spp. to develop live vaccine strains. The Salmonella Typhimurium, ST454 strain (ST WT) was irradiated with Cobalt60 gamma-irradiator at 1.5 kGy for 1 h to maximize the mutation rate, and attenuated daughter colonies were screened using in vitro macrophage replication capacity and in vivo mouse infection assays. Among 30 candidates, ATOMSal-L6, with 9,961-fold lower virulence than the parent strain (ST454) in the mouse LD50 model, was chosen. This vaccine candidate was mutated at 71 sites, and in particular, lost one bacteriophage. As a vaccine, ATOMSal-L6 induced a Salmonella-specific IgG response to provide effective protective immunity upon intramuscular vaccination of mice. Furthermore, when mice and sows were orally immunized with ATOMSal-L6, we found a strong protective immune response, including multifunctional cellular immunity. These results indicate that ATOMSal-L6 is the first live vaccine candidate to be developed using R-MET, to the best of our knowledge. R-MET can be used as a fast and effective live vaccine development technology that can be used to develop vaccine strains against emerging or serotype-shifting pathogens.

Structural and Biochemical Characterization of Thioredoxin-2 from Deinococcus radiodurans.

Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H2O2 treated conditions. Both drtrx1 and drtrx2 genes were induced following H2O2 treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H2O2, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

Radiation-Inactivated S. gallinarum Vaccine Provides a High Protective Immune Response by Activating Both Humoral and Cellular Immunity.

Salmonella enterica subsp. enterica serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.

Protective Effect of Polysaccharides Extracted from Cudrania tricuspidata Fruit against Cisplatin-Induced Cytotoxicity in Macrophages and a Mouse Model.

Although cisplatin is one of most effective chemotherapeutic drugs that is widely used to treat various types of cancer, it can cause undesirable damage in immune cells and normal tissue because of its strong cytotoxicity and non-selectivity. This study was conducted to investigate the cytoprotective effects of Cudrania tricuspidata fruit-derived polysaccharides (CTPS) against cisplatin-induced cytotoxicity in macrophages, lung cancer cell lines, and a mouse model, and to explore the possibility of application of CTPS as a supplement for anticancer therapy. Both cisplatin alone and cisplatin with CTPS induced a significant cytotoxicity in A549 and H460 lung cancer cells, whereas cytotoxicity was suppressed by CTPS in cisplatin-treated RAW264.7 cells. CTPS significantly attenuated the apoptotic and necrotic population, as well as cell penetration in cisplatin-treated RAW264.7 cells, which ultimately inhibited the upregulation of Bcl-2-associated X protein (Bax), cytosolic cytochrome c, poly (adenosine diphosphateribose) polymerase (PARP) cleavage, and caspases-3, -8, and -9, and the downregulation of B cell lymphoma-2 (Bcl-2). The CTPS-induced cytoprotective action was mediated with a reduction in reactive oxygen species production and mitochondrial transmembrane potential loss in cisplatin-treated RAW264.7 cells. In agreement with the results obtained above, CTPS induced the attenuation of cell damage in cisplatin-treated bone marrow-derived macrophages (primary cells). In in vivo studies, CTPS significantly inhibited metastatic colonies and bodyweight loss as well as immunotoxicity in splenic T cells compared to the cisplatin-treated group in lung metastasis-induced mice. Furthermore, CTPS decreased the level of CRE and BUN in serum. In summation, these results suggest that CTPS-induced cytoprotective action may play a role in alleviating the side effects induced by chemotherapeutic drugs.

Chrysin Derivative CM1 and Exhibited Anti-Inflammatory Action by Upregulating Toll-Interacting Protein Expression in Lipopolysaccharide-Stimulated RAW264.7 Macrophage Cells.

Although our previous study revealed that gamma-irradiated chrysin enhanced anti-inflammatory activity compared to intact chrysin, it remains unclear whether the chrysin derivative, CM1, produced by gamma irradiation, negatively regulates toll-like receptor (TLR) signaling. In this study, we investigated the molecular basis for the downregulation of TLR4 signal transduction by CM1 in macrophages. We initially determined the appropriate concentration of CM1 and found no cellular toxicity below 2 µg/mL. Upon stimulation with lipopolysaccharide (LPS), CM1 modulated LPS-stimulated inflammatory action by suppressing the release of proinflammatory mediators (cytokines TNF-α and IL-6) and nitric oxide (NO) and downregulated the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, CM1 markedly elevated the expression of the TLR negative regulator toll-interacting protein (Tollip) in dose- and time-dependent manners. LPS-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II), proinflammatory cytokines (TNF-α and IL-6), COX-2, and iNOS-mediated NO were inhibited by CM1; these effects were prevented by the knockdown of Tollip expression. Additionally, CM1 did not affect the downregulation of LPS-induced expression of MAPKs and NF-κB signaling in Tollip-downregulated cells. These findings provide insight into effective therapeutic intervention of inflammatory disease by increasing the understanding of the negative regulation of TLR signaling induced by CM1.

Immunoregulatory properties of a crude extraction fraction rich in polysaccharide from Chrysanthemum zawadskii Herbich var. latilobum and its potential role as a vaccine adjuvant.

The objective of the current study was to demonstrate the immunostimulatory effects of a polysaccharide isolated from Chrysanthemum zawadskii Herbich var. latilobum leaves (CP) and evaluate its potential as a vaccine adjuvant. Results showed that CP induced maturation of the dendritic cells (DCs). In addition, CP-treated DCs activated naïve T cells to polarized CD4+ and CD8+ T cells and substantially induced the production of IFN-γ and IL-2 in vitro. Furthermore, CP initiated the maturation of DCs via the activation of MAPK and NF-κB signaling pathways. Interestingly, systemic administration of CP-treated DCs pulsed with ovalbumin (OVA) peptides significantly enhanced the immune response in vivo, which included the generation of antigen (OVA)-specific polyfunctional T cells, increased cytotoxic T lymphocyte activity, induction of Th1-mediated humoral immunity, and suppression of tumor growth. Taken together, our study highlighted the immunoregulatory activity of CP as well as its potential as a candidate vaccine adjuvant.

Bombyx batryticatus Protein-Rich Extract Induces Maturation of Dendritic Cells and Th1 Polarization: A Potential Immunological Adjuvant for Cancer Vaccine.

Bombyx batryticatus, a protein-rich edible insect, is widely used as a traditional medicine in China. Several pharmacological studies have reported the anticancer activity of B. batryticatus extracts; however, the capacity of B. batryticatus extracts as immune potentiators for increasing the efficacy of cancer immunotherapy is still unverified. In the present study, we investigated the immunomodulatory role of B. batryticatus protein-rich extract (BBPE) in bone marrow-derived dendritic cells (BMDCs) and DC vaccine-immunized mice. BBPE-treated BMDCs displayed characteristics of mature immune status, including high expression of surface molecules (CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II), increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukin-12p70), enhanced antigen-presenting ability, and reduced endocytosis. BBPE-treated BMDCs promoted naive CD4+ and CD8+ T-cell proliferation and activation. Furthermore, BBPE/ovalbumin (OVA)-pulsed DC-immunized mice showed a stronger OVA-specific multifunctional T-cell response in CD4+ and CD8+ T cells and a stronger Th1 antibody response than mice receiving differently treated DCs, which showed the enhanced protective effect against tumor growth in E.G7 tumor-bearing mice. Our data demonstrate that BBPE can be a novel immune potentiator for a DC-based vaccine in anticancer therapy.

Comparison of Exosomes Derived from Non- and Gamma-Irradiated Melanoma Cancer Cells as a Potential Antigenic and Immunogenic Source for Dendritic Cell-Based Immunotherapeutic Vaccine.

Cancer cells can secrete exosomes under various stressful conditions, whose functions are involved in the delivery of various biologically active materials into host cells and/or modulation of host immune responses. Therefore, an improved understanding of the immunological interventions that stress-induced tumor exosomes have may provide novel therapeutic approaches and more effective vaccine designs. Here, we confirmed the phenotypical and functional alterations of dendritic cells (DCs), which act as a bridge between the innate and adaptive arms of immunity, following non-irradiated (N-exo) and gamma-irradiated melanoma cancer cell-derived exosome (G-exo) stimulation, and evaluated the N-exo- and G-exo-stimulated DCs as therapeutic cancer vaccine candidates. We demonstrated that G-exo-stimulated DCs result in DC maturation by the upregulation of surface molecule expression, pro-inflammatory cytokine release, and antigen-presenting ability, and the downregulation of endocytic capacity. In addition, these cells promoted T cell proliferation and the generation of T helper type 1 (Th1) and interferon (IFN)-γ-producing CD8+ T cells. However, N-exo-stimulated DCs induced semi-mature phenotypes and functions, eventually inhibiting T cell proliferation, decreasing IFN-γ, and increasing IL-10-producing CD4+ T cells. In addition, although N-exo and G-exo stimulations showed similar levels of antigen-specific IFN-γ production, which served as tumor antigen sources in melanoma-specific T cells, G-exo-stimulated DC vaccination conferred a stronger tumor growth inhibition than N-exo-stimulated DC vaccination; further, this was accompanied by a high frequency of tumor-specific, multifunctional effector T cells. These results suggest that gamma irradiation could provide important clues for designing and developing effective exosome vaccines that can induce strong immunogenicity, especially tumor-specific multifunctional T cell responses.

Edible Oxya chinensis sinuosa-Derived Protein as a Potential Nutraceutical for Anticancer Immunity Improvement.

Oxya chinensis sinuosa (Ocs) is consumed as representative edible insects in Asia, but its function in various immune systems remains unclear. This study aimed to demonstrate the immunomodulatory effect, particularly on the innate and adaptive immune response, of Ocs protein (Ocs-P) and to investigate its function as a potent anticancer immunostimulant when administered during the progression stage of colon carcinoma in tumor-bearing mice. Our in vitro results demonstrated that Ocs-P treatment induces phenotypic alteration (increased expression of surface molecules and production of Th1-polarizing cytokines and decreased antigen uptake ability) of dendritic cells (DCs) through the activation of MAPK and NF-κB-dependent signaling pathways. Additionally, Ocs-P-stimulated DCs initiated differentiation of naive T cells into IFN-γ-producing Th1-type T cells effectively and activated cytotoxic CD8+ T cell response. In colon carcinoma-bearing mouse models, oral administration of Ocs-P inhibited tumor growth and restored the expression of decreased surface molecules in lineage-CD11c+MHC-II+ splenic DCs. Furthermore, Ocs-P administration enhanced the generation of multifunctional CD4+ and CD8+ T cells expressing Th1-type cytokines (TNF-α, IFN-γ, and IL-2) and the degranulation marker (CD107a). Collectively, these results suggest that Ocs-P demonstrates an immunostimulatory effect and may induce powerful anticancer immunity.

Irradiation-Induced Intestinal Damage Is Recovered by the Indigenous Gut Bacteria Lactobacillus acidophilus.

The intestinal tract is one of the most sensitive organs following irradiation. The protective effect of specific indigenous microbiota on irradiation-induced damage to intestinal epithelial cells has not been reported. Mice were irradiated with a single dose of 6 Gy of gamma rays. The intestinal damage was analyzed by histopathology. Intestinal stemness and differentiation were determined by intestinal organoid culture. Microbiota community was observed by high-throughput 16S rRNA gene sequencing and oligotyping analysis. We showed that distal small intestine was damaged by sublethal dose of gamma irradiation. Intestinal organoids derived from the irradiated mice showed defects in budding and mucin expression, suggesting the detrimental effect of irradiation on the intestinal stemness and differentiation. In addition, irradiation reduced intestinal immunoglobulin A level, concomitant with decreased microbiota diversity based on our high-throughput 16S rRNA gene sequencing data. Especially, the relative abundance of Lactobacillus was reduced at early time point post-irradiation; however, it was recovered at late time point. Oligotyping analysis within the Lactobacillus genus indicated that Lactobacillus-related oligotype 1 (OT1) including Lactobacillus acidophilus might drive recovery after irradiation as it was associated with increased long-term numbers post-exposure. We showed that treatment with heat-killed L. acidophilus rescued the budding-impaired organoids and induced sufficient differentiation in epithelial cells, and particularly mucin-producing cells, in intestinal organoids. This study provides the first evidence that the indigenous gut bacteria L. acidophilus enhance intestinal epithelial function with respect to irradiation-induced intestinal damage by improving intestinal stem cell function and cell differentiation.

Development of Oxytolerant Salmonella typhimurium Using Radiation Mutation Technology (RMT) for Cancer Therapy.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Antioxidant Activities of an Exopolysaccharide (DeinoPol) Produced by the Extreme Radiation-Resistant Bacterium Deinococcus radiodurans.

Deinococcus radiodurans shows extreme resistance to a range of remarkable environmental stresses. Deinococcal exopolysaccharide (DeinoPol) is a component of the cell wall, but its role in stress resistance has not yet been well-described. In this study, we isolated and characterized DeinoPol from Deinococcus radiodurans R1 strain and investigated its application as an antioxidant agent. Bioinformatic analysis indicated that dra0033, encoding an ExoP-like protein, was involved in DeinoPol biosynthesis, and dra0033 mutation significantly decreased survival rates in response to stresses. Purified DeinoPol consists of different monosaccharides and has a molecular weight of approximately 80 to 100 kDa. DeinoPol also demonstrates highly protective effects on human keratinocytes in response to stress-induced apoptosis by effectively scavenging ROS. Taken together, these findings indicate that DeinoPol is the first reported deinococcal exopolysaccharide that might be used in cosmetics and pharmaceuticals as a safe and attractive radical scavenger.

Crystal structure of the highly radiation-inducible DinB/YfiT superfamily protein DR0053 from Deinococcus radiodurans R1.

Deinococcus radiodurans is an extremophilic bacterium well-known for its extraordinary resistance to ionizing radiation and other DNA damage- and oxidative stress-generating agents. In addition to its efficient DNA damage repair and oxidative stress resistance mechanisms, protein family expansions and stress-induced genes/proteins are also regarded as important components that add to the robustness of this bacterium. D. radiodurans encodes specific expansions of 13 DinB/YfiT homologs, which is a relatively large number when compared to those found in Gram-positive bacteria. In this study, we investigated the expression profiles of 13 dinB genes after γ-irradiation, mitomycin C and H2O2 treatment. dr0053 had the highest expression levels after DNA-damage inducing γ-irradiation and MMC treatment, increasing ∼200-fold and ∼16-fold, respectively. We also determined the crystal structure of DR0053 at 2.07 Šresolution. DR0053 adopted a typical four-helix bundle structure that is characteristic of DinB/YfiT proteins. A putative metal binding site was occupied by zinc even though the highly conserved His triad of DinB/YfiT proteins was replaced by Glu-Asn-His.

Gamma-irradiation-killed Streptococcus pneumoniae potently induces the expression of IL-6 and IL-8 in human bronchial epithelial cells.

Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been developed and its immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immunity, in the present study, we investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cell-line, BEAS-2B, by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP). r-SP potently induced interleukin (IL)-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner, whereas h-SP and f-SP poorly induced them. Of note, the mRNA levels of IL-6 and IL-8 were approximately two-fold higher when cells were stimulated with 3 × 107 CFU/ml of r-SP for 3 h, while the protein levels of IL-6 and IL-8 were approximately five-fold higher after stimulation with 3 × 107 CFU/ml of r-SP for 24 h. Furthermore, r-SP exhibited potent activation of Toll-like receptor 2 compared with h-SP or f-SP. The expression of IL-6 and IL-8 induced by r-SP was mediated through the activation of mitogen-activated protein kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the aforementioned activities. Taken together, we suggest that r-SP stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8, which might influence the induction of adaptive immune responses.

Gamma-irradiation of Streptococcus pneumoniae for the use as an immunogenic whole cell vaccine.

Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation method to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r-SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.