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

Psychological factors that promote behavior modification by obese patients

<p>Abstract</p> <p>Background</p> <p>The weight-loss effect of team medical care in which counseling is provided by clinical psychologists was investigated in an university hospital obesity (OB) clinic. Nutritional and exercise therapy were also studied. In our previous study, we conducted a randomized, controlled trial with obese patients and confirmed that subjects who received counseling lost significantly more weight than those in a non-counseling group. The purpose of this study was to identify the psychological characteristics assessed by ego states that promote behavior modification by obese patients.</p> <p>Methods</p> <p>147 obese patients (116 females, 31 males; mean age: 45.9 ± 15.4 years) participated in a 6-month weight-loss program in our OB clinic. Their psychosocial characteristics were assessed using the Tokyo University Egogram (TEG) before and after intervention. The Wilcoxon signed rank test was used to compare weight and psychological factors before and after intervention. Multiple regression analysis was used to identify factors affecting weight loss.</p> <p>Results</p> <p>Overall, 101 subjects (68.7%) completed the program, and their data was analyzed. The subjects mean weight loss was 6.2 ± 7.3 kg (<it>Z </it>= 7.72, <it>p </it>< 0.01), and their mean BMI decreased by 2.4 ± 2.7 kg/m²(<it>Z </it>= 7.65, <it>p </it>< 0.01). Significant differences were observed for the Adult (A) ego state (0.68 ± 3.56, <it>Z </it>= 1.95, <it>p </it>< 0.05) and the Free Child (FC) ego state (0.59 ± 2.74, <it>Z </it>= 2.46, <it>p </it>< 0.01). The pre-FC ego state had a significant effect on weight loss (β = 0.33, <it>p </it>< 0.01), and a tendency for changes in the A ego state scores to affect weight loss (β = - 0.20, <it>p </it>= 0.06) was observed.</p> <p>Conclusion</p> <p>This study of a 6-month weight-loss program that included counseling by clinical psychologists confirmed that the A ego state of obese patients, which is related to their self-monitoring skill, and the FC ego state of them, which is related to their autonomy, were increased. Furthermore, the negative aspects of the FC ego state related to optimistic and instinctive characteristics inhibited the behavior modification, while the A ego state represented objective self-monitoring skills that may have contributed to weight loss.</p

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)

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

Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression

We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG)3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.ArticleFEBS Open Bio. 3:41-45 (2013)journal articl

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice

Background: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. Methods: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). Results: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1 alpha and IL-6 in the colon compared to a vector control strain. Conclusions: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.ArticleMICROBIAL CELL FACTORIES. 14:189 (2015)journal articl

Pretreatment serum FGF-23 levels predict the efficacy of calcitriol therapy in dialysis patients

Pretreatment serum FGF-23 levels predict the efficacy of calcitriol therapy in dialysis patients.BackgroundThe predictor for the result of calcitriol therapy would be useful in the clinical practice of secondary hyperparathyroidism. Fibroblast growth factor-23 (FGF-23) is a newly found circulating phosphaturic factor. Its circulating level is elevated in uremia.MethodsDialysis patients with plasma intact parathyroid hormone (iPTH) levels greater than 300 pg/mL were included in the study. Calcitriol was intravenously injected three times a week. The patients whose plasma iPTH levels dropped below 300 pg/mL within 24 weeks were defined as those who had been successfully treated. A sandwich enzyme-linked immunosorbent assay (ELISA) system that detects human FGF-23 was applied.ResultsSixty-two patients were analyzed. The pretreatment FGF-23 levels were related to the iPTH levels, calcium × phosphate product levels, and history of active vitamin D therapy. The pretreatment FGF-23, iPTH, and calcium levels were lower in the patients who would be successfully treated with calcitriol. A logistic regression study revealed that the pretreatment iPTH and FGF-23 levels significantly affected the therapy results. Analyses using a receiver-operated curve revealed that FGF-23 was the best screening test for identifying patients with future refractory response to calcitriol therapy. The treatment would be successful in 88.2% of those with FGF-23 ≤9860 ng/L and iPTH ≤591 pg/mL, while it would be successful in only 4.2% of those with FGF-23 >9860 ng/L and iPTH >591 pg/mL.ConclusionPretreatment serum FGF-23 levels were a good indicator in predicting the response to calcitriol therapy. The measurement of serum FGF-23 levels, especially in combination with iPTH levels, is a promising laboratory examination for the clinical practice of secondary hyperparathyroidism

Inhibitory/Suppressive oligodeoxynucleotide nanocapsules as simple oral delivery devices for preventing atopic dermatitis in mice

Advance online publication 6 January 2015Here, we report a simple and low-cost oral oligodeoxynucleotide (ODN) delivery system targeted to the gut Peyer's patches (PPs). This system requires only Dulbecco's modified eagle's medium, calcium chloride, ODNs, and basic laboratory equipment. ODN nanocapsules (ODNcaps) were directly delivered to the PPs through oral administration and were taken up by macrophages in the PPs, where they induced an immune response. Long-term continuous oral dosing with inhibitory/suppressive ODNcaps (iODNcaps, "iSG3caps" in this study) was evaluated using an atopic dermatitis mouse model to visually monitor disease course. Administration of iSG3caps improved skin lesions and decreased epidermal thickness. Underlying this effect is the ability of iSG3 to bind to and prevent phosphorylation of signal transducer and activator of transcription 6, thereby blocking the interleukin-4 signaling cascade mediated by binding of allergens to type 2 helper T cells. The results of our iSG3cap oral delivery experiments suggest that iSG3 may be useful for treating allergic diseases.ArticleMOLECULAR THERAPY. 23(2):297-309 (2015)journal articl

Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from -Lactoglobulin Secreted by Lactococcus lactis

Previous studies showed that hydrolysates of -lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus.The study was supported by a grant from the Iijima Memorial Foundation for the Promotion of Food Science and Technology to Takeshi Shimosato