Development of a New Treatment Modality for Lung Diseases That Uses Innovative Fine Droplet Drying (fdd) Technology Offering Inhalable Nano/microparticles-incorporated Therapeutic Agents.

ArticleImpact. 2019(3): 56-58. (2019)journal articl

Intratracheally Therapeutic Option for COPD: A Potential Usage of the Therapeutic Microbe for Delivering Specific Protein to the Lungs

Currently, inhaled therapy using corticosteroids and/or bronchodilators is the major established treatment for chronic obstructive pulmonary disease (COPD). The topic to be covered in this chapter is the recently developed experimental approach using biologically active molecules secreted by the live genetically modified lactic acid bacteria (gmLAB). The strategy to use gmLAB as a therapeutic/delivering tool targeting disease-specific active molecules/cites is proceeding. The role of inflammation and oxidative stress in COPD development is a valid target point. Heme oxygenase (HO)-1 as an anti-inflammatory and antioxidative stress molecule has been examined to attenuate the lung function decline and inflammation in the murine model of COPD. Recently, HO-1-secreting gmLAB as a tool for targeting inflammatory diseases has been developed and examined in several disease models including COPD. When administered intratracheally, the gmLAB showed migration to the peripheral lung and overexpression of anti-inflammatory/oxidative HO-1 in both lung and serum, protecting the lung from COPD development

Expression of the immunoreactive buckwheat major allergenic storage protein in Lactococcus lactis

Proteins from buckwheat (Fagopyrum esculentum) are strong allergens that can cause serious symptoms, including anaphylaxis, in patients with hypersensitivity. In this study, we successfully developed a modified lactic acid bacterial vector (pNSH) and a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced a major allergenic storage protein of buckwheat, Fagag1 (61.2 kDa, GenBank accession number AF152003), with or without a green fluorescent protein (GFP) tag. GFP fluorescence allows for rapid, simple, and accurate measurement of target protein expression by microscopy or fluorimetry. We describe a convenient method for production of rGFP-Fagag1 fusion and rFagag1 proteins with a good yield in an advantageous probiotic host. We found that in vitro treatment of splenocytes isolated from buckwheat crude protein-immunized mice with rFagag1 increased the expression of allergic inflammation cytokines such as IL-4, IL-13, and IL-17 F. Because it was less antigenic, rGFP-Fagag1 protein from NZ9000 might be of limited use; however, rFagag1 from NZ9000 evoked a robust response as measured by induction of IL-4 and IL-17 F expression levels. The observed allergic activity is indicative of a Th2 cell-mediated immune response and is similar to the effects induced by exposure to buckwheat crude protein. Our results suggest that expression of rFagag1 in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to buckwheat allergens.ArticleAPPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 97(8):3603-3611 (2013)journal articl

Expression of a Biologically Active GFP-alpha(S1)-Casein Fusion Protein in Lactococcus lactis

In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to alpha(S1)-casein (GFP-alpha(S1)Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and alpha(S1)-casein, a major cow's milk allergen, and the resulting vector, pNZ8148#2-GFP-alpha(S1)Cas, was applied to the expression of recombinant GFP-alpha(S1)Cas protein (rGFP-alpha(S1)Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-alpha(S1)Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from alpha-casein (a parts per thousand yen70 % alpha(S)-casein)-immunized mice with rGFP-alpha(S1)Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune response and is similar to the effects induced by exposure to alpha-casein. Our results suggest that the expression of rGFP-alpha(S1)Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to specific milk allergen.ArticleCURRENT MICROBIOLOGY. 64(6):569-575 (2012)journal articl

Parallel evolution of dominant pistil-side self-incompatibility suppressors in Arabidopsis

Selfing is a frequent evolutionary trend in angiosperms, and is a suitable model for studying the recurrent patterns underlying adaptive evolution. Many plants avoid self-fertilization by physiological processes referred to as self-incompatibility (SI). In the Brassicaceae, direct and specific interactions between the male ligand SP11/SCR and the female receptor kinase SRK are required for the SI response. Although Arabidopsis thaliana acquired autogamy through loss of these genes, molecular evolution contributed to the spread of self-compatibility alleles requires further investigation. We show here that in this species, dominant SRK silencing genes have evolved at least twice. Different inverted repeat sequences were found in the relic SRK region of the Col-0 and C24 strains. Both types of inverted repeats suppress the functional SRK sequence in a dominant fashion with different target specificities. It is possible that these dominant suppressors of SI contributed to the rapid fixation of self-compatibility in A. thaliana

CpG oligodeoxynucleotides induce strong up-regulation of interleukin 33 via Toll-like receptor 9

We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24 h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1 alpha, IL-1 beta and IL-18, are down-regulated after 24 h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods.ArticleBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 394(1):81-86 (2010)journal articl

Secretion of an immunoreactive single-chain variable fragment antibody against mouse interleukin 6 by Lactococcus lactis

Epub 2016 Oct 8.Interleukin 6 (IL-6) is an important pathogenic factor in development of various inflammatory and autoimmune diseases and cancer. Blocking antibodies against molecules associated with IL-6/IL-6 receptor signaling are an attractive candidate for the prevention or therapy of these diseases. In this study, we developed a genetically modified strain of Lactococcus lactis secreting a single-chain variable fragment antibody against mouse IL-6 (IL6scFv). An IL6scFv-secretion vector was constructed by cloning an IL6scFv gene fragment into a lactococcal secretion plasmid and was electroporated into L. lactis NZ9000 (NZ-IL6scFv). Secretion of recombinant IL6scFv (rIL6scFv) by nisin-induced NZ-IL6scFv was confirmed by western blotting and was optimized by tuning culture conditions. We found that rIL6scFv could bind to commercial recombinant mouse IL-6. This result clearly demonstrated the immunoreactivity of rIL6scFv. This is the first study to engineer a genetically modified strain of lactic acid bacteria (gmLAB) that produces a functional anti-cytokine scFv. Numerous previous studies suggested that mucosal delivery of biomedical proteins using gmLAB is an effective and low-cost way to treat various disorders. Therefore, NZ-IL6scFv may be an attractive tool for the research and development of new IL-6 targeting agents for various inflammatory and autoimmune diseases as well as for cancer.ArticleAPPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 101(1):341-349 (2017)journal articl

Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZ

Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.ArticleANIMAL SCIENCE JOURNAL. 80(5):597-604 (2009)journal articl

Synergistic oligodeoxynucleotide strongly promotes CpG-induced interleukin-6 production

[Background] :Bacterial genomes span a significant portion of diversity, reflecting their adaptation strategies; these strategies include nucleotide usage biases that affect chromosome configuration. Here, we explore an immuno-synergistic oligodeoxynucleotide (iSN-ODN, named iSN34), derived from Lactobacillus rhamnosusGG (LGG) genomic sequences, that exhibits a synergistic effect on immune response to CpG-induced immune activation. [Methods]: The sequence of iSN34 was designed based on the genomic sequences of LGG. Pathogen-free mice were purchased from Japan SLC and maintained under temperature- and light-controlled conditions. We tested the effects of iSN34 exposure in vitro and in vivo by assessing effects on mRNA expression, protein levels, and cell type in murine splenocytes. [Results]: We demonstrate that iSN34 has a significant stimulatory effect when administered in combination with CpGODN, yielding enhanced interleukin (IL)-6 expression and production. IL-6 is a pleotropic cytokine that has been shown to prevent epithelial apoptosis during prolonged inflammation. [Conclusions]: Our results are the first report of a bacterial-DNA-derived ODN that exhibits immune synergistic activity.The potent over-expression of IL-6 in response to treatment with the combination of CpG ODN and iSN34 suggests anew approach to immune therapy.This finding may lead to novel clinical strategies for the prevention or treatment of dysfunctions of the innate and adaptive immune systems.This work was supported by A-STEP (Adaptable and Seamless Technology Transfer Program through Target-driven R&D)