miR103a-3p in extracellular vesicles from FcεRI-aggregated human mast cells enhances IL-5 production by group 2 innate lymphoid cells.

BACKGROUND: Mast cells (MCs) are key regulators of IgE-mediated allergic inflammation. Cell-derived extracellular vesicles (EVs) contain bioactive compounds such as microRNAs. EVs can transfer signals to recipient cells, thus using a novel mechanism of cell-to-cell communication. However, whether MC-derived EVs are involved in FcεRI-mediated allergic inflammation is unclear. OBJECTIVE: We sought to investigate the effect of EVs derived from FcεRI-aggregated human MCs on the function of human group 2 innate lymphoid cells (ILC2s). METHODS: Human cultured MCs were sensitized with and without IgE for 1 hour and then incubated with anti-IgE antibody, IL-33, or medium alone for 24 hours. EVs in the MC supernatant were isolated by using ExoQuick-TC. RESULTS: Coculture of ILC2s with EVs derived from the FcεRI-aggregated MCs significantly enhanced IL-5 production and sustained upregulation of IL-5 mRNA expression in IL-33-stimulated ILC2s, but IL-13 production and IL-13 mRNA expression were unchanged. miR103a-3p expression was upregulated in IL-33-stimulated ILC2s that had been cocultured with EVs derived from anti-IgE antibody-stimulated MCs. Transduction of an miR103a-3p mimic to ILC2s significantly enhanced IL-5 production by IL-33-stimulated ILC2s. miR103a-3p promoted demethylation of an arginine residue of GATA3 by downregulating protein arginine methyltransferase 5 (PRMT5) mRNA. Reduction of protein arginine methyltransferase 5 expression in ILC2s by using a small interfering RNA technique resulted in upregulation of IL-5 production by IL-33-stimulated ILC2s. Furthermore, the level of miR103a-3p expression was significantly higher in EVs from sera of patients with atopic dermatitis than in EVs from nonatopic healthy control subjects. CONCLUSION: Eosinophilic allergic inflammation may be exacerbated owing to ILC2 activation by MC-derived miR103a-3p.

A modified perlite protocol with a mixed dimethyl sulfoxide and trehalose cryoprotectant improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.

Homolka's perlite protocol (HPP) was evaluated for cryopreservation of a wide range of ectomycorrhizal basidiomycete cultures, then a modified perlite protocol (MPP), in which cryoprotectant was added just before freezing rather than during the culturing process, was applied to cryosensitive strains that failed to survive when HPP was used. Further modifications of MPP with various cryoprotectants were explored to improve the cryopreservation of hard-to-preserve strains. The efficacy of HPP was assessed in 111 strains of 38 species of basidiomycetes of various cryosensitivities. After freezing strains using HPP, the viability and colony diameter of the strains were examined after 2 wk, 6 mo, and 1 y of storage at -80 C. Of the 111 strains tested, 91 survived after 1 y of storage with high viability of 80% or more, whereas the remaining 20 strains exhibited low and unstable viability. For those selected cryosensitive strains that did not survive well when HPP was used, MPP was applied with a mixture of cryoprotectants, dimethyl sulfoxide (DMSO), glycerol, and trehalose, at different concentrations and combinations. Toxicity testing of the cryoprotectants in the nonfrozen state revealed that 12% (v/v) glycerol was highly toxic for six strains (four species), whereas DMSO (5% and 10% [v/v]) was less toxic than glycerol. The viability of the cryosensitive strains after freezing demonstrated that DMSO was more efficient than glycerol, and trehalose enhanced the cryoprotective effects of both glycerol and DMSO when MPP was used for cryopreservation. Our comparative analysis of MPP with various combinations and concentrations of cryoprotectants revealed that a mixture of 5% DMSO and 10% trehalose was the most effective cryoprotectant, and that using MPP with this cryoprotectant was applicable to many cryosensitive strains.

Cyclic analogue of S-benzylisothiourea that suppresses kynurenine production without inhibiting indoleamine 2,3-dioxygenase activity.

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC50 = 0.34 µM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment.

Characterization of human decidual mast cells and establishment of a culture system.

BACKGROUND: Although rodent decidual mast cells (MCs) reportedly play an important role in implantation and placenta formation, the characterization of human decidual MCs has been not well clarified. The aims of this study were to investigate the distribution and characteristics of MCs in human decidua and to establish a culture system for decidua-derived MCs. METHODS: Decidual tissues were obtained from patients who underwent a legal elective abortion (6th week to 9th week of pregnancy), and decidual MCs were enzymatically dispersed. Cultured decidua-derived MCs were generated by culturing decidual cells with stem cell factor. An ultrastructural analysis of primary decidual MCs and cultured decidua-derived MCs was performed using a transmission electron microscope. Receptor and protease expression was analyzed using FACS. Histamine released from MCs was measured using enzyme immune assays. RESULTS: A larger proportion of tryptase positive(+) MCs in decidua was present on the maternal side. Both enzymatically dispersed decidual MCs and cultured decidua-derived MCs showed an FcεRIα+Kit+tryptase+chymase+ phenotype. Their granules contenting particles exhibited variable amounts of electron-lucent space separating electron-dense particles. Both enzymatically dispersed decidual MCs and cultured decidua-derived MCs released comparable amounts of histamine following FcεRI aggregation. CONCLUSIONS: The isolation method for MCs from decidua during early pregnancy and the culture system for decidua-derived MCs may enable the roles of decidual MC during pregnancy to be explored.

Activation of human synovial mast cells from rheumatoid arthritis or osteoarthritis patients in response to aggregated IgG through Fcγ receptor I and Fcγ receptor II.

OBJECTIVE: Substantial evidence suggests that human synovial mast cells (MCs) are involved in the pathogenesis of rheumatoid arthritis (RA). A plausible pathway for the activation of synovial MCs is through IgG receptors, given the prevalence of circulating IgG isotype autoantibodies and synovial immune complexes in patients with RA. However, IgG receptor expression on human synovial MCs remains uncharacterized. The aim of this study was to identify which IgG receptor(s) on synovial MCs are responsible for MC activation in immune complexes. METHODS: Synovial tissue specimens were obtained from patients with RA or patients with osteoarthritis (OA) who were undergoing joint replacement surgery, and synovial MCs were enzymatically dispersed. Cultured synovium-derived MCs were generated by culturing synovial cells with stem cell factor, and receptor expression was analyzed using fluorescence-activated cell sorting. Mediators released from MCs were measured using enzyme immunoassays or enzyme-linked immunosorbent assays. RESULTS: Primary synovial MCs and cultured synovium-derived MCs obtained from both patients with RA and patients with OA expressed Fcε receptor I (FcεRI), FcγRI, and FcγRII but not FcγRIII. Cultured synovium-derived MCs induced degranulation and the production of prostaglandin D2 and tumor necrosis factor α (TNFα) through FcγRI. The aggregation of FcγRII caused histamine release from cultured MCs but not from primary MCs. Histamine release induced by aggregated IgG was significantly inhibited by neutralizing anti-FcγRI monoclonal antibody and anti-FcγRII monoclonal antibody. CONCLUSION: With regard to the FcR expression profile, synovial MCs from patients with RA and patients with OA were similar. FcγRI was responsible for producing abundant TNFα from synovial MCs in response to aggregated IgG. Immune complexes may activate synovial MCs through FcγRI and FcγRII.

Development of a recombinant adenovirus vector production system free of replication-competent adenovirus by utilizing a packaging size limit of the viral genome.

In a conventional adenovirus (Ad) vector production method using 293 cells, homologous recombination between Ad vector DNA and 293 cell-derived Ad E1 DNA occurs with low efficiency, resulting in the generation of replication-competent adenovirus (RCA). RCA can induce the spread of replication-incompetent Ad vectors, leading to unexpected tissue damage. In order to overcome this problem, we developed an Ad vector production system free of RCA generation by utilizing the Ad packaging size limit of the viral genome. It is well known that up to approximately 105% (37.7 kb) of the wild-type genome (35.9 kb) can be packaged in the Ad virion. We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit. In accord with our strategy, no RCA generation was observed during the passages when we used the E1 (3.2kb)-deleted Ad vectors containing a more than 3.0-kb transgene expression cassette in the E3 region. In contrast, the E1 (3.2kb)-deleted Ad vectors, which retain 37.7 kb of the viral genome and have an insertion of a 2.1-kb transgene expression cassette in the E3 region, generated RCA, although RCA derived from this Ad vector exceeded the packaging size limit (105.0%). These results suggest that RCA generation can be avoided when the genome size of RCA is more than 108.3% (38.9 kb) of the wild-type Ad genome. This Ad vector production system generates safe, easy, and efficient Ad vector stock for both basic study as well as clinical research.

Activation of human mast cells through the platelet-activating factor receptor.

BACKGROUND: In human subjects platelet-activating factor (PAF) concentrations are markedly increased in the plasma after anaphylactic reactions, and these correlate strongly with the severity of the response. The mechanism for the systemic spread of mast cell (MC) activation in anaphylaxis is often assumed to relate to the hematogenous spread of allergen, but this is implausible, and amplification mechanisms need to be considered. OBJECTIVE: We have investigated the ability of PAF to induce human MC degranulation using skin, lung, and peripheral blood (PB)-derived cultured MCs and the signaling pathways activated in PB-derived MCs in response to PAF. METHODS: The expression of PAF receptor was investigated by means of RT-PCR and Western blot analysis. Cell-signaling pathways in PB-derived MCs in response to PAF were investigated by analyzing the effect of various inhibitors and the silencing of phospholipase C (PLC) mRNA on PAF-mediated histamine release. RESULTS: We show for the first time that PAF induces histamine release from human lung MCs and PB-derived MCs but not skin MCs. Activation of PAF receptor-coupled G(alphai) leads to degranulation through PLCgamma1 and PLCbeta2 activation in human MCs. PAF-induced degranulation was rapid, being maximal at 5 seconds, and was partially dependent on extracellular Ca(2+). CONCLUSION: Our findings provide a mechanism whereby PAF mediates an amplification loop for MC activation in the generation of anaphylaxis.

Role of MyD88 and TLR9 in the innate immune response elicited by serotype 5 adenoviral vectors.

A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.

Comparison of gene expression efficiency and innate immune response induced by Ad vector and lipoplex.

Vectors for gene expression are the essential tools for both gene therapy and basic research. There are two groups of gene therapy vectors, viral and non-viral vectors. At present, toxicity triggered by vectors is one of the major concerns for clinical trials. In general, non-viral vectors, such as plasmid DNA-cationic liposome complex (lipoplex), are thought to be safer than viral vectors, such as adenovirus (Ad) vector, although lipoplex is less efficient in term of gene expression than the Ad vector. However, there has been no study directly comparing the gene expression efficiency and safety of viral and non-viral vectors. Here, we present evidence that the Ad vector shows much more efficient gene expression and is safer than lipoplex, at least with respect to the innate immune response. After being systemically administered to mice, the Ad vector showed a transduction efficiency that was 2 to 5 log orders higher than that of lipoplex, depending on the organ. On the other hand, surprisingly, the administration of lipoplex produced a greater amount of inflammatory cytokines such as interleukin-6, interleukin-12, and tumor necrosis factor-alpha than did the administration of the Ad vector, whereas a comparable level of hepatotoxicity was induced by these vectors. The production of inflammatory cytokines induced by the injection of lipoplex was reduced when the CpG motifs were removed completely from plasmid DNA. Thus, care should be taken to ensure the innate immune response induced by gene therapy vectors, especially lipoplex.

Fiber-modified adenovirus vectors decrease liver toxicity through reduced IL-6 production.

Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c(+) cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses.

Fiber-modified adenovirus vectors mediate efficient gene transfer into undifferentiated and adipogenic-differentiated human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are considered a source of cells for regenerative medicine, and cell and gene therapy. Efficient gene transfer into hMSCs is essential for basic investigations into cellular differentiation and developmental biology, and for therapeutic applications in gene-modified regenerative medicine. In the present study, we optimized the transduction of hMSCs by means of fiber-modified adenovirus (Ad) vectors. Among the various types of Ad vectors tested, the polylysine modification of the C-terminal of the fiber knob most markedly improved the efficiency of hMSC transduction. At 300 vector particles per cell of polylysine-modified Ad vectors, more than 95% of the hMSCs expressed transgene. In this condition, polylysine-modified Ad vectors mediated 460-fold more transgene activity than the conventional Ad vectors. Ad vectors containing the Ad type 35 fiber or an Arg-Gly-Asp (RGD) peptide in the fiber knob mediated 130 or 16 times, respectively, the transgene activity mediated by the conventional Ad vectors. We also examined the efficiency of transduction into adipogenic-differentiated hMSCs. In this latter case, only Ad vectors containing the Ad type 35 fiber showed efficient gene expression. These results showed that fiber-modified Ad vectors could become a potent tool for basic research into, and the therapeutic application of, hMSCs and adipogenic-differentiated hMSCs.

A new heat shock gene, AgsA, which encodes a small chaperone involved in suppressing protein aggregation in Salmonella enterica serovar typhimurium.

We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Here we show that overproduction of AgsA partially complements the DeltadnaK52 thermosensitive phenotype and reduces the amount of heat-aggregated proteins in both DeltadnaK52 and DeltarpoH mutants of Escherichia coli. These data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures.