Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/ Ribavirin Therapy in Chronic Hepatitis C

Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3− NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed.　Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.     

On-Chip Immunoelectrophoresis of Extracellular Vesicles Released from Human Breast Cancer Cells

Extracellular vesicles (EVs) including exosomes and microvesicles have attracted considerable attention in the fields of cell biology and medicine. For a better understanding of EVs and further exploration of their applications, the development of analytical methods for biological nanovesicles has been required. In particular, considering the heterogeneity of EVs, methods capable of measuring individual vesicles are desired. Here, we report that on-chip immunoelectrophoresis can provide a useful method for the differential protein expression profiling of individual EVs. Electrophoresis experiments were performed on EVs collected from the culture supernatant of MDA-MB-231 human breast cancer cells using a measurement platform comprising a microcapillary electrophoresis chip and a laser dark-field microimaging system. The zeta potential distribution of EVs that reacted with an anti-human CD63 (exosome and microvesicle marker) antibody showed a marked positive shift as compared with that for the normal immunoglobulin G (IgG) isotype control. Thus, on-chip immunoelectrophoresis could sensitively detect the over-expression of CD63 glycoproteins on EVs. Moreover, to explore the applicability of on-chip immunoelectrophoresis to cancer diagnosis, EVs collected from the blood of a mouse tumor model were analyzed by this method. By comparing the zeta potential distributions of EVs after their immunochemical reaction with normal IgG, and the anti-human CD63 and anti-human CD44 (cancer stem cell marker) antibodies, EVs of tumor origin circulating in blood were differentially detected in the real sample. The result indicates that the present method is potentially applicable to liquid biopsy, a promising approach to the low-invasive diagnosis of cancer.     

Computer-Aided Prediction of Long-Term Prognosis of Patients with Ulcerative Colitis after Cytoapheresis Therapy

Cytoapheresis (CAP) therapy is widely used in ulcerative colitis (UC) patients with moderate to severe activity in Japan. The aim of this study is to predict the need of operation after CAP therapy of UC patients on an individual level using an artificial neural network system (ANN). Ninety UC patients with moderate to severe activity were treated with CAP. Data on the patients’ demographics, medication, clinical activity index (CAI) and efficacy of CAP were collected. Clinical data were divided into training data group and validation data group and analyzed using ANN to predict individual outcomes. The sensitivity and specificity of predictive expression by ANN were 0.96 and 0.97, respectively. Events of admission, operation, and use of immunomodulator, and efficacy of CAP were significantly correlated to the outcome. Requirement of operation after CAP therapy was successfully predicted by using ANN. This newly established ANN strategy would be used as powerful support of physicians in the clinical practice.     

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.     

Laminin receptor mediates anti-inflammatory and anti-thrombogenic effects of pigment epithelium-derived factor in myeloma cells

Pigment epithelium-derived factor (PEDF) has anti-inflammatory and anti-thrombogenic properties both in cell culture and animal models. Although adipose triglyceride lipase (ATGL) and laminin receptor (LR) are two putative receptors for PEDF, which receptor mainly mediates the beneficial effects of PEDF is largely unknown. In this study, we addressed the issue. siRNA raised against LR (siLR) and siATGL transfection dramatically decreased LR and ATGL levels in human cultured myeloma cells, respectively. Ten nM PEDF significantly reduced vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels in siCon- or siATGL-transfected myeloma cells, whereas PEDF increased rather than decreased these gene expressions in siLR-transfected cells. Neutralizing antibody directed against LR (LR-Ab) or LR antagonist actually bound to LR and reduced mRNA levels of VEGF, MCP-1, ICAM-1 and PAI-1 in myeloma cells. Further, pre-treatment of LR-Ab or LR antagonist suppressed the binding of PEDF to LR and resultantly blocked the effects of PEDF in myeloma cells. In addition, high concentration of LR agonist mimicked the actions of PEDF on these gene expressions in myeloma cells. This study indicates that PEDF causes anti-angiogenic, anti-inflammatory and anti-thrombogenic reactions in myeloma cells through the interaction with LR. Target domain of LR agonist and antagonist might be involved in the PEDF-signaling to gene suppression in myeloma cells.     

Genetic structure and putative selective sweep in the pioneer tree,Zanthoxylum ailanthoides

Zanthoxylum ailanthoides Siebold & Zucc. is one of the most frequently encountered pioneer trees in Japanese warm–temperate evergreen oak forests. Our previous study in one region of Japan suggested high levels of population differentiation and putative natural selection acting on one of the nuclear loci analyzed. Here, we extend our analysis to study the genetic structure of 10 populations of Z. ailanthoides across Japan using 9 simple sequence repeat (SSR) loci for a better understanding of its genetic structure. First, the southernmost population (Kagoshima) in the samples was found to have the highest genetic diversity, suggesting there was a glacial refugium at or near the location of the population. Second, relatively strong genetic differentiation was found among populations, and there was a positive correlation between genetic distances and geographic distances (Mantel test; P < 0.001). Based on this information, we analyzed nucleotide variation at the putatively selected locus homologous to the gene encoding the ADP-glucose pyrophosphorylase large subunit (agpL). Despite the strong genetic differentiation among populations suggested by the SSR loci, the agpL locus was monomorphic in almost all populations analyzed. The results of this study strongly supported the possibility of a selective sweep at or near the agpL locus.     

Discovery of imidazo[1,2-b]pyridazine derivatives as IKKβ inhibitors. Part 1: Hit-to-lead study and structure–activity relationship

Imidazo[1,2-b]pyridazine derivatives from high-throughput screening were developed as IKKβ inhibitors. By the optimization of the 3- and 6-position of imidazo[1,2-b]pyridazine scaffold, cell-free IKKβ inhibitory activity and TNFα inhibitory activity in THP-1 cell increased. Also, these compounds showed high kinase selectivity. The structure–activity relationship was revealed and the interaction model of imidazo[1,2-b]pyridazine compounds with IKKβ was constructed.     

Efficient synthesis of 6-<Emphasis Type="Italic">O</Emphasis>-palmitoyl-1,2-<Emphasis Type="Italic">O</Emphasis>-isopropylidene-α-<Emphasis Type="SmallCaps">d</Emphasis>-glucofuranose in an organic solvent system by lipase-catalyzed esterification

In order to synthesize a sugar ester at high concentration, 1,2-O-isopropylidene-α-d-glucofuranose (IpGlc), which is one of the sugar acetals and is more hydrophobic than unmodified glucose, was esterified with palmitic acid at 40°C using immobilized lipase from Candida antarctica in some organic solvents or their mixtures. Acetone + t-butyl alcohol (3:1 v/v) improved both the initial reaction rate and yield after 80 h: the product reached its maximum value (240 mmol/kg solvent; ca. 110 g/kg solvent) when 400 mmol IpGlc/kg solvent and 1,200 mmol palmitic acid/kg solvent were used in this solvent mixture.     

Nifedipine, a calcium channel blocker, inhibits inflammatory and fibrogenic gene expressions in advanced glycation end product (AGE)-exposed fibroblasts via mineralocorticoid receptor antagonistic activity

Advanced glycation end products (AGE) are involved in tissue damage and remodeling. This study investigated whether AGE could elicit inflammatory and fibrogenic reactions in fibroblast cell line MRC-5 cells via autocrine production of aldosterone and if nifedipine could block the AGE actions through mineralocorticoid receptor (MR) antagonistic activity. AGE significantly up-regulated monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-β (TGF-β), type III collagen and receptor for AGE (RAGE) mRNA levels in MRC-5 cells, all of which were completely blocked by nifedipine or an MR antagonist spironolactone. Aldosterone also dose-dependently increased MCP-1, TGF-β and type III collagen mRNA levels in MRC-5 cells, which were suppressed by nifedipine, but not amlodipine, a control calcium channel blocker. Further, AGE significantly stimulated aldosterone generation in MRC-5 cells, which was partially blocked by nifedipine or spironolactone. In this study, we demonstrated for the first time that AGE could evoke inflammatory and fibrogenic reactions in MRC-5 cells via aldosterone production, which were blocked by the MR antagonistic activity of nifedipine. Our present study provides a unique beneficial aspect of nifedipine on tissue damage and remodeling; it could work as an anti-inflammatory and anti-fibrogenic agent against AGE via MR antagonistic activity.