Expression of B subunit of E. coli heat-labile enterotoxin in the progenies of transgenic tobacco bred by crossing nuclear- and chloroplast-transgenic lines.

The B subunit of Escherichia coli heat-labile toxin (LTB) is a model antigen that induces a strong immune response upon oral administration and enhances immune responses to conjugated and co-administered antigens. We previously examined high expression levels of LTB in plants by chloroplast and synthetic LTB gene expression and found substantially higher expression levels of LTB, compared to nuclear LTB expression in wild-type plants. The 2.5% LTB protein of total soluble protein that was observed by chloroplast transformation was approximately 250-fold greater expression than that of LTB via nuclear genome integration. In addition, the amount of LTB protein found in transgenic tobacco leaves using a synthetic LTB gene was 2.2% of the total soluble plant protein, which was approximately 200-fold higher than that in plants with native LTB gene expression. The purpose of our experiment was to increase LTB levels in plants by crossing chloroplast-transformed and synthetic LTB transgenic lines produced previously to express higher LTB levels. LTB protein levels in the F1 transgenic tobacco plants was significantly higher (3.3%), compared to the 2.2% of chloroplast-transformed line or 2.8% of synthetic LTB gene line. Our results suggest that LTB expression was successfully enhanced in the F1 hybrid generation of transgenic tobacco plants.

Electrorheological response of inorganic-coated multi-wall carbon nanotubes with core-shell nanostructure.

The effect of coating thickness and sequence on the multi-wall carbon nanotube (MWCNT) surface on electrorheological (ER) activity is investigated. Silica-coated MWCNTs with different shell thicknesses and inorganic-coated MWCNTs with different shell sequences are fabricated using the sol-gel process. The morphology and elemental analyses of the core-shell structured MWCNTs were performed and the effect of coating thickness and coating materials on the MWCNT surface on ER and dielectric properties of inorganic-coated MWCNT suspensions have been analyzed from the measurement results of shear stress, viscosity, current density and permeability. The electrical conductivity of silica-coated MWCNTs decreases with an increase of the shell thickness. However, the dynamic yield stress and viscoelastic properties under an external electric field increased when the shell thickness reached 20 nm and then decreased with the thickness up to 40 nm. However, the titania-coated MWCNT suspension with a shell thickness of 40 nm showed the highest dynamic yield stress compared to the other core-shell structured MWCNT suspension at the same volume fraction. It has been found that the material of the outermost shell plays an important role in the ER performance. It has been concluded that the electrical conductivity and the permittivity of the MWCNTs can be controlled by adjusting the coating thickness and sequence of inorganic materials.

NLRP3 Inflammasome as Therapeutic Targets in Inflammatory Diseases.

Innate immunity is a first line defence system in the body which is for sensing signals of danger such as pathogenic microbes or host-derived signals of cellular stress. Pattern recognition receptors (PRR's), which present in the cell memebrane, are suspect the infection through pathogen-associated molecular patterns (PAMP), and activate innate immunity with response to promote inflammation via inflammatory cells such as macrophages and neutrophils, and cytokines. Inflammasome are protein complexes which are part of innate immunity in inflammation to remove pathogens and repair damaged tissues. What is the important role of inflammation in disease? In this review, we are focused on the action mechanism of NLRP3 inflammasome in inflammatory diseases such as asthma, atopic dermatitis, and sepsis.

Effectiveness of the Novel Herbal Medicine, KIOM-MA, and Its Bioconversion Product, KIOM-MA128, on the Treatment of Atopic Dermatitis.

This study was conducted to determine if oral administration of the novel herbal medicine, KIOM-MA, and its Lactobacillus acidophilus-fermented product, KIOM-MA128, has therapeutic properties for the treatment of atopic dermatitis (AD). Using AD-induced BALB/c mice by Ovalbumin and aluminum hydroxide, the effectiveness of KIOM-MA and KIOM-MA128 on AD was evaluated. Oral administration of KIOM-MA and KIOM-MA128 reduced major clinical signs of AD including erythema/darkening, edema/papulation, excoriations, lichenification/prurigo, and dryness. Interestingly, KIOM-MA128 more significantly improved AD-related symptoms including decrease of IgE level in the plasma as well as reduction of scratching behavior, skin severity in the AD BALB/c model. HPLC analysis showed the significant changes in the constituent patterns between KIOM-MA and KIOM-MA128. Our results suggest that both KIOM-MA and KIOM-MA128 have potential for therapeutic reagent for the treatment of AD, and further, the efficacy is significantly enhanced by L. acidophilus fermentation via increases in its indicator molecule.

The n-Hexane, ethylacetate, and butanol fractions from Hydnocarpi Semen enhanced wound healing in a mice ulcer model.

Our previous report showed that Hydnocarpi Semen (HS) extract has wound repair activity at ulcer lesion in diabetic mice. In this study, fractions of n-Hexane, ethylacetate (EtOAc), and butanol (BuOH) from HS crude extract were evaluated for their wound healing activity by using in vivo diabetic ulcer models and in vitro acute inflammation model. Although n-Hexane and EtOAc fractions promote wound healing in mice with ulcer, the BuOH fraction exhibited the most potent wound healing activity and the wound area score significantly decreased after treatment of BuOH fraction even at dose of 2 mg/kg. BuOH fraction stimulated macrophages to increase the production of nitric oxide (NO) and TNF-α. The BuOH fraction also enhanced the production of TGF-ß and VEGF, which were involved in fibroblast activation and angiogenesis. The mRNA expression and activation of MMP-9 were increased by three fractions and the activity was higher in BuOH fraction-treated group compared to the other groups. The mechanism that the HS helps to promote healing of diabetic ulcer is possibly associated with the production of TNF-α, a proinflammatory cytokine, as well as the secretion of VEGF, TGF-ß, and MMP-9, which were involved in proliferation of capillaries and fibroblasts. These results suggest that HS can be a new candidate material for the treatment of wound in skin ulcer.

An essential role for the antiviral endoribonuclease, RNase-L, in antibacterial immunity.

Type I IFNs were discovered as the primary antiviral cytokines and are now known to serve critical functions in host defense against bacterial pathogens. Accordingly, established mediators of IFN antiviral activity may mediate previously unrecognized antibacterial functions. RNase-L is the terminal component of an RNA decay pathway that is an important mediator of IFN-induced antiviral activity. Here, we identify a role for RNase-L in the host antibacterial response. RNase-L(-/-) mice exhibited a dramatic increase in mortality after challenge with Bacillus anthracis and Escherichia coli; this increased susceptibility was due to a compromised immune response resulting in increased bacterial load. Investigation of the mechanisms of RNase-L antibacterial activity indicated that RNase-L is required for the optimal induction of proinflammatory cytokines that play essential roles in host defense from bacterial pathogens. RNase-L also regulated the expression of the endolysosomal protease, cathepsin-E, and endosome-associated activities, that function to eliminate internalized bacteria and may contribute to RNase-L antimicrobial action. Our results reveal a unique role for RNase-L in the antibacterial response that is mediated through multiple mechanisms. As a regulator of fundamental components of the innate immune response, RNase-L represents a viable therapeutic target to augment host defense against diverse microbial pathogens.

Cell Autonomous Circadian Systems and Their Relation to Inflammation.

All living beings on earth have an important mechanism of 24-h periodicity, which controls their physiology, metabolism, and behavior. In humans, 24-h periodicity is regulated by the superchiasmatic nucleus (SCN) through external and environmental cues. Peripheral organs demonstrate circadian rhythms and circadian clock functions, and these are also observed in cultured cell lines. Every cell contains a CLOCK: BMAL1 loop for the generation of circadian rhythms. In this review, we focused on cell autonomous circadian rhythms in immune cells, the inflammatory diseases caused by disruption of circadian rhythms in hormones, and the role of clock genes in inflammatory diseases.

Comparison of two mice strains, A/J and C57BL/6, in caspase-1 activity and IL-1beta secretion of macrophage to Mycobacterium leprae infection.

A/J mice were found to have amino acid differences in Naip5, one of the NOD-like receptors (NLRs) involved in the cytosolic recognition of pathogen-associated molecular patterns and one of the adaptor proteins for caspase-1 activation. This defect was associated with a susceptibility to Legionella infection, suggesting an important role for Naip5 in the immune response also to other intracellular pathogens, such as Mycobacterium leprae. In this study, the immune responses of macrophages from A/J mice against M. leprae were compared to those of macrophages from C57BL/6 mice. Infection with M. leprae induced high levels of TNF-alpha production and NF-kappaB activation in A/J and C57BL/6 macrophages. Caspase-1 activation and IL-1beta secretion were also induced in both macrophages. However, macrophages from A/J mice exhibited reduced caspase-1 activation and IL-1beta secretion compared to C57BL/6 macrophages. These results suggest that NLR family proteins may have a role in the innate immune response to M. leprae.

Repositioned Drugs for Inflammatory Diseases such as Sepsis, Asthma, and Atopic Dermatitis.

The process of drug discovery and drug development consumes billions of dollars to bring a new drug to the market. Drug development is time consuming and sometimes, the failure rates are high. Thus, the pharmaceutical industry is looking for a better option for new drug discovery. Drug repositioning is a good alternative technology that has demonstrated many advantages over de novo drug development, the most important one being shorter drug development timelines. In the last two decades, drug repositioning has made tremendous impact on drug development technologies. In this review, we focus on the recent advances in drug repositioning technologies and discuss the repositioned drugs used for inflammatory diseases such as sepsis, asthma, and atopic dermatitis.

Anti-tumor activity of oxypeucedanin from Ostericum koreanum against human prostate carcinoma DU145 cells.

PURPOSE: Oxypeucedanin has been reported to have various biological activities. We investigated the efficacy of a coumarin compound, oxypeucedanin, from Ostericum koreanum against the human prostate carcinoma cell line DU145. MATERIAL AND METHODS: Oxypeucedanin (C(16)H(14)O(5), mw: 286) was isolated through silica gel chromatography and characterized by NMR. The cells were treated with oxypeucedanin (25, 50, and 100 microM) for 24-72 hours, and cell growth and death were then assayed. The cell cycle progression and apoptotic effects were also assessed by western blotting. RESULTS: Treatment with oxypeucedanin inhibited cell growth and induced cell death in DU145 cells. Furthermore, oxypeucedanin-induced cell growth inhibition was associated with an increase in G2-M arrest in cell cycle progression in DU145 cells in a dose and time-dependent manner. G2-M arrest by oxypeucedanin was associated with decreased levels of cyclin A, cyclin B1, Cdc2, and pCdc2. Oxypeucedanin-induced cell death was associated with significant increases in apoptosis and cleaved caspase-3 and poly-(ADP-ribose) polymerase. CONCLUSION: These finding suggest a novel anticancer effect for oxypeucedanin, mediated via induction of G2-M cell cycle arrest and apoptosis in human prostate carcinoma DU145 cells.

Expression of sialyltransferase activity on intact human neutrophils.

Endogenous polymorphonuclear leukocyte (PMN)-associated sialidase activity enhances PMN adhesion to and migration across the endothelium through the removal of sialylated cell-surface residues. We tested the hypothesis that PMNs also express sialyltransferase (ST) activity that restores sialyl residues to the PMN surface. We developed a highly sensitive fluorometric assay to demonstrate that intact human PMNs can mediate and accept sialyl residue transfer. This ST activity is inhibited by a ST inhibitor, CMP, which also inhibits the transendothelial migration of PMNs in response to IL-8 in vitro and in vivo. We conclude that intact PMNs express sialidase and ST activities that permit rapid modulation of their surface sialylation and their ability to adhere to and migrate across the endothelium.

Development of an Equine Antitoxin by Immunizing the Halla Horse with the Receptor-Binding Domain of Botulinum Neurotoxin Type A1.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, 20 µL of the week 24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international unit (IU) of a reference equine antitoxin. Furthermore, 20 µL of equine serum and 100 µg of purified equine F(ab')2 showed 100% neutralization of 10,000 LD50 in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.

Expression of cholera toxin B subunit in transgenic rice endosperm.

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Mechanical modeling of self-expandable stent fabricated using braiding technology.

The mechanical behavior of a stent is one of the important factors involved in ensuring its opening within arterial conduits. This study aimed to develop a mechanical model for designing self-expandable stents fabricated using braiding technology. For this purpose, a finite element model was constructed by developing a preprocessing program for the three-dimensional geometrical modeling of the braiding structure inside stents, and validated for various stents with different braiding structures. The constituent wires (Nitinol) in the braided stents were assumed to be superelastic material and their mechanical behavior was incorporated into the finite element software through a user material subroutine (VUMAT in ABAQUS) employing a one-dimensional superelastic model. For the verification of the model, several braided stents were manufactured using an automated braiding machine and characterized focusing on their compressive behavior. It was observed that the braided stents showed a hysteresis between their loading and unloading behavior when a compressive load was applied to the braided tube. Through the finite element analysis, it was concluded that the current mechanical model can appropriately predict the mechanical behavior of braided stents including such hysteretic behavior, and that the hysteresis was caused by the slippage between the constituent wires and their superelastic property.

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Rifampicin Alleviates Atopic Dermatitis-Like Response in vivo and in vitro.

Atopic dermatitis (AD) is a common inflammatory skin disorder mediated by inflammatory cells, such as macrophages and mast cells. Rifampicin is mainly used for the treatment of tuberculosis. Recently, it was reported that rifampicin has anti-inflammatory and immune-suppressive activities. In this study, we investigated the effect of rifampicin on atopic dermatitis in vivo and in vitro. AD was induced by treatment with 2, 4-dinitrochlorobenzene (DNCB) in NC/Nga mice. A subset of mice was then treated with rifampicin by oral administration. The severity score and scratching behavior were alleviated in the rifampicin-treated group. Serum immunoglobulin E (IgE) and interleukin-4 (IL-4) levels were also ameliorated in mice treated with rifampicin. We next examined whether rifampicin has anti-atopic activity via suppression of mast cell activation. Rifampicin suppressed the release of ß-hexosaminidase and histamine from human mast cell (HMC)-1 cultures stimulated with compound 48/80. Treatment with rifampicin also inhibited secretion of inflammatory mediators, such tumor necrosis factor-α (TNF-α) and prostaglandin D2 (PGD2), in mast cells activated by compound 48/80. The mRNA expression of cyclooxygenase 2 (COX-2) was reduced in the cells treated with rifampicin in a concentration-dependent manner. These results suggest that rifampicin can be used to treat atopic dermatitis.

A novel staphylococcal enterotoxin B subunit vaccine candidate elicits protective immune response in a mouse model.

Staphylococcal enterotoxin B (SEB), produced by the gram-positive bacterium Staphylococcus aureus, is responsible for food poisoning and toxic shock syndrome, and is considered a potential bioterrorism agent. Unfortunately, still now no approved vaccines are available against SEB. In this study, we constructed a series of nontoxic SEB mutants (mSEBs) and examined whether these mSEBs provide protective immunity against SEB challenge. These mSEB vaccine candidates did not demonstrate superantigen activity in mouse splenocyte cultures. Immunization with the vaccine candidates triggered the production of IgG-antibodies with neutralizing activity. In addition, increased production of IgG1 and IgG3 was observed after immunization, which signifies both Th1- and Th2-induced immune responses. Among the vaccine candidates tested, S9, a double mutant (N23A and Y90A) and S19, a quadruple mutant (N23A, Y90A, R110A, and F177A), demonstrated complete protection against a lethal SEB challenge. Altogether, our results strongly suggest that these mSEBs could be an effective recombinant SEB vaccine candidates for further/future preclinical and clinical studies.

A role for Stat1 in the regulation of lipopolysaccharide-induced interleukin-1beta expression.

Because the induction of interleukin-1beta (IL-1beta) is critical to antibacterial host defenses and its excessive generation is a prominent component of sepsis, regulation of this proinflammatory cytokine is a critical factor in the immune response to lipopolysaccharide (LPS). We previously showed that LPS-induced IL-1beta expression was regulated by a Stat1-dependent, nitric oxide (NO)-mediated mechanism. Subsequent in vivo studies showed that whereas Stat1 had a role in the downregulation of IL-1beta expression, it had a more significant effect on its initial induction. Although both interferon-beta (IFN-beta) and IFN-gamma activate Stat1, the early appearance of IFN-beta in the circulation after LPS administration suggested its pivotal role in Stat1-mediated IL-1beta expression in vivo. Further in vitro analysis of peritoneal macrophages from IFN-beta (/), Stat1(/), and caspase-1(/) mice and their wild-type controls following LPS stimulation demonstrated that IL-1beta mRNA was expressed in these mice but not in macrophages from MyD88(/) mice. Despite the presence of IL-1beta mRNA, IL-1beta protein was markedly reduced in the absence of Stat1 activation in macrophages derived from IFN-beta (/) and Stat1(/) mice or in the absence of caspase-1 activity, which itself was dependent on Stat1 activation. These studies support the hypothesis that the expression of IL-1beta requires both the MyD88-dependent induction of IL-1beta mRNA and pro-IL-1beta as well as the MyD88-independent, Stat1-mediated processing of that gene product into active cytokine.

Expression of a synthetic cholera toxin B subunit in tobacco using ubiquitin promoter and bar gene as a selectable marker.

A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin (PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT.