Tsunao Kishida

Involvement of IL-17A in the pathogenesis of DSS-induced colitis in mice

To investigate the etiological implication of IL-17A in inflammatory bowel disease (IBD), dextran sodium sulfate (DSS) was administered to the mice deficient for the IL-17A gene. They showed only faint manifestations of colitis, as revealed by body weight loss, shrinkage in the colon length, serum haptoglobin concentration, and disease activity index. Although the mortality rate of WT mice reached approximately 60%, more than 90% of the IL-17A KO mice survived the DSS treatment. Histological change was also marginal in the IL-17A KO intestine, in which epithelial damage and inflammatory infiltrates were not obvious and the myeloperoxidase activity elevated only slightly. G-CSF and MCP-1 were abundantly produced in WT mouse intestine, whereas the production of these chemokines was drastically hampered in IL-17A-null intestine. The present results show that IL-17A plays a pivotal role in the pathogenesis of DSS-induced colitis, while MCP-1 and G-CSF may be crucially involved in the IL-17A-induced inflammation.

Raspberry-like assembly of cross-linked nanogels for protein delivery.

Raspberry-like assembly of nanogels (A-CHPNG) with a high potential as a carrier for protein delivery was prepared. Cross-linking of acrylate group-modified cholesterol-bearing pullulan nanogel (CHPANG) with thiol group-modified poly (ethylene glycol) (PEGSH) by Michael addition yielded A-CHPNG with narrow size distribution. The size of A-CHPNGs was controlled in the range of 40-120 nm by changing the concentration of CHPANG and PEGSH. A-CHPNG gradually degraded by hydrolysis under physiological condition and seemed to dissociate back to original nanogel. A-CHPNG encapsulated interleukin-12 (IL-12) efficiently (96%) and stably kept it in the presence of BSA (50 mg/ml). In addition, A-CHPNG had a high potential to maintain a high IL-12 level in plasma after subcutaneous injection in mice. Therefore, A-CHPNG is a promising carrier for long-term medications.

Interleukin-27 Activates Natural Killer Cells and Suppresses NK-Resistant Head and Neck Squamous Cell Carcinoma through Inducing Antibody-Dependent Cellular Cytotoxicity

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fcgamma receptor III-dependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.

N-acetylcysteine prevents nitric oxide-induced chondrocyte apoptosis and cartilage degeneration in an experimental model of osteoarthritis.

We investigated whether N‐acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)‐induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0–2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase‐3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO‐induced apoptosis, ROS overproduction, p53 up‐regulation, and caspase‐3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis‐preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO‐induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.

IL-21 Administration into the Nostril Alleviates Murine Allergic Rhinitis

Type I allergic diseases such as allergic rhinitis are caused by IgE-mediated humoral immune responses, while eosinophils also fulfill important roles in the etiology of IgE-mediated allergy. IL-21 regulates growth, differentiation, and function of T, B, and NK cells, while the production of IgE is also influenced by IL-21. In this study we examined whether IL-21 is capable of controlling IgE-mediated allergic reactions in vivo by using the allergic rhinitis mouse model that was established by repetitive sensitization and intranasal challenge with OVA. Intranasal administration with recombinant mouse IL-21 (rmIL-21) significantly reduced the number of sneezes, as well as the serum concentration of OVA-specific IgE, in comparison with that of untreated allergic mice. The rmIL-21 treatment also suppressed germline Cε transcription in the nasal-associated lymphoid tissues, which may have, at least partly, resulted from the up-regulation of Bcl-6 mRNA caused by IL-21. Local expression of IL-4, IL-5, and IL-13 was also inhibited by the intranasal cytokine therapy whereas, in contrast, the expression of endogenous IL-21 mRNA was induced by exogenous rmIL-21. Moreover, IL-21 acted on nasal fibroblasts to inhibit production of eotaxin. This novel function of IL-21 may be associated with the attenuation of eosinophil infiltration into nasal mucosa that was revealed by histopathological observation. These results indicated that IL-21 nasal administration effectively ameliorated allergic rhinitis through pleiotropic activities, i.e., the prevention of IgE production by B cells and eotaxin production by fibroblasts.

Intra-Articular Injection of Hyaluronan Restores the Aberrant Expression of Matrix Metalloproteinase-13 in Osteoarthritic Subchondral Bone

Subchondral bone is a candidate for treatment of osteoarthritis (OA). We investigated the effects of intra‐articular injection of hyaluronan (IAI‐HA) on subchondral bone in rabbit OA model. OA was induced by anterior cruciate ligament transection, with some rabbits receiving IAI‐HA. OA was graded morphologically, and expression of mRNA was assessed by real‐time RT‐PCR. Tissue sections were stained with hyaluronan‐binding protein, and penetration of fluorescent hyaluronan was assessed. The in vitro inhibitory effect of hyaluronan on MMP‐13 was analyzed in human osteoarthritic subchondral bone osteoblasts (OA Ob) by real‐time RT‐PCR and ELISA. Binding of hyaluronan to OA Ob via CD44 was assessed by immunofluorescence cytochemistry. Expression of MMP‐13 and IL‐6 mRNA in cartilage and subchondral bone, and morphological OA grade, increased over time. IAI‐HA ameliorated the OA grade and selectively suppressed MMP‐13 mRNA in subchondral bone. IAI‐HA enhanced the hyaluronan staining of subchondral bone marrow cells and osteocyte lacunae. Fluorescence was observed in the subchondral bone marrow space. In OA Ob, hyaluronan reduced the expression and production of MMP‐13, and anti‐CD44 antibody blocked hyaluronan binding to OA Ob. These findings indicate that regulation of MMP‐13 in subchondral bone may be a critical mechanism during IAI‐HA.

Early-stage blocking of Notch signaling inhibits the depletion of goblet cells in dextran sodium sulfate-induced colitis in mice

BackgroundGoblet cells, which contribute to mucosal defense and repair in the intestinal epithelium, are depleted in human and rodent colitis. The Notch signal pathway regulates the differentiation of intestinal stem cells into epithelial cells and inhibits the differentiation of secretory lineages, including goblet cells. The aim of our study was to clarify whether the blocking of the Notch pathway at an early stage of colitis would preserve goblet cells and facilitate the healing process in dextran sulfate sodium (DSS)-induced colitis in mice.MethodsDSS was orally administered to C57/BL6 mice for 7xa0days, and dibenzazepine (DBZ), a Notch pathway blocker, was administered for 5 consecutive days, beginning on the first day of DSS treatment. Colonic mucosal inflammation was evaluated clinically, biochemically, and histologically. The expression of the goblet cell-associated genes Math1 and MUC2 and proinflammatory cytokines was evaluated by real-time reverse-transcriptase-PCR, with the expression of Math1 and MUC2 also visualized by immunohistochemical examination.ResultsThe administration of DBZ at 4xa0μmol/kg significantly reduced the severity of the colitis. Compared with the DSS only-treated intestine, the number of goblet cells was relatively sustained, and the expression of Math1 and MUC2 was also elevated in the DSS/DBZ-treated intestine. DBZ treatment suppressed the mRNA levels for interleukin-1β and -6, and matrix metalloproteinases-3 and -9 in the DSS-treated intestine.ConclusionsEarly-stage blocking of Notch signaling may ameliorate acute DSS colitis by preventing reduction in the number of goblet cells.

Comparison of anti-rheumatic effects of local RNAi-based therapy in collagen induced arthritis rats using various cytokine genes as molecular targets.

RNA interference (RNAi) provides a powerful means of sequence-specific gene silencing. Several studies show that RNAi may provide promising strategies to treat human diseases by suppressing disease responsible genes in vivo. In locomotor diseases, the progression of collagen-induced arthritis (CIA) is suppressed by tumor necrosis factor-α (TNF-α)-specific small interfering RNA (siRNA) delivered into the joint. The aim of this study, is to compare the effects of intraarticularly administered siRNAs targeting TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and receptor activator of NF-κB ligand (RANKL) on CIA in rats. We confirmed that the silencing effects of siRNA duplexes specific for rat TNF-α, IL-1β, IL-6 and RANKL in vitro. Each siRNA was also delivered into the knee joint of CIA rats by the in vivo electroporation method 7, 10, 13 and 16xa0days after immunization with collagen. Local delivery of TNF-α or IL-1β-specific siRNA ameliorated CIA in rats effectively at the gross morphological, radiographical and histological evaluations. Our results suggested that TNF-α and IL-1β were the cytokines to be targeted in the joint for the treatment of rheumatoid arthritis. The in vivo siRNA transfection method may be useful for selection of target molecules to be silenced for treatment of joint diseases.

MDR1a/1b gene silencing enhances drug sensitivity in rat fibroblast-like synoviocytes.

Drug resistance mediated by P‐glycoprotein (P‐gp) is one of the major reasons for the failure of rheumatoid arthritis (RA) therapy with disease modifying anti‐rheumatic drugs and glucocorticoids. In the present study, we aimed to investigate the in vitro effectiveness of small interfering RNA (siRNA) to render rat fibroblast‐like synoviocytes (FLS) susceptible to drugs. We also attempted the electroporation‐mediated transfer of siRNA against multidrug resistance (MDR) genes into rat knee joints.

Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination.

BACKGROUNDnIn melanoma patients vaccinated with monocyte-derived melanoma peptide-pulsed dendritic cells (DC), the delayed-type hypersensitivity (DTH) reactions have been examined as a surrogate marker to determine if acquired immunity is induced by DC vaccination. To date, however, only limited information has been reported as for histopathological analyses of DTH.nnnOBJECTIVEnTo evaluate tumor-specific immunomonitoring histopathologically after DC vaccination in melanoma patients.nnnMETHODSnSeven patients previously vaccinated with monocyte-derived melanoma peptide-pulsed DCs were challenged with recall antigenic peptide injection in the skin of the forearm. Using immunohistochemical techniques, the presence of immune cells and the expression of CD4, CD8, interleukin (IL)-2, IL-4, IL-10, Foxp3, CD1a, CD1d, and interferon (IFN)-gamma was investigated at the site of injection where a DTH reaction developed.nnnRESULTSnStrong DTH reactions from infiltrated erythema to bullae formation were detected in all 7 cases. Biopsies taken from the DTH site revealed heavy infiltration of mononuclear cells and eosinophils in the dermis and subcutaneous tissue. Cells staining positively for CD4, CD8, IL-2, IL-4, Foxp3, CD1d, and IFN-gamma were increased at the site 48h after antigen injection in all cases. Cells positive for IL-10 were never found in any patient. Regulatory T cells appeared 6h after injection and reached their maximum at day 7.nnnCONCLUSIONSnThe significant induction of CD8(+)T cells as well as both Th1 and Th2-type cells at the site of DTH suggests that effective antigen presentation leading to anti-tumor immune responses has taken place. Inhibitory mechanisms may also develop as the disappearance of the DTH response could be related to an increase in Foxp3+ cells.