Shu-Fen Wu

Antiviral Effects of an Iminosugar Derivative on Flavivirus Infections

ABSTRACT Endoplasmic reticulum (ER) α-glucosidase inhibitors, which block the trimming step of N-linked glycosylation, have been shown to eliminate the production of several ER-budding viruses. Here we investigated the effects of one such inhibitor, N-nonyl-deoxynojirimycin (NN-DNJ), a 9-carbon alkyl iminosugar derivative, on infection by Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2). In the presence of NN-DNJ, JEV and DEN-2 infections were suppressed in a dose-dependent manner. This inhibitory effect appeared to influence DEN-2 infection more than JEV infection, since lower concentrations of NN-DNJ substantially blocked DEN-2 replication. Secretion of the flaviviral glycoproteins E and NS1 was greatly reduced, and levels of DEN-2 viral RNA replication measured by fluorogenic reverse transcription-PCR were also decreased, by NN-DNJ. Notably, the viral glycoproteins, prM, E, and NS1 were found to associate transiently with the ER chaperone calnexin, and this interaction was affected by NN-DNJ, suggesting a potential role of calnexin in the folding of flaviviral glycoproteins. Additionally, in a mouse model of lethal challenge by JEV infection, oral delivery of NN-DNJ reduced the mortality rate. These findings show that NN-DNJ has an antiviral effect on flavivirus infection, likely through interference with virus replication at the posttranslational modification level, occurring mainly in the ER.

Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice.

To evaluate the potential of DNA vaccine against dengue (DEN) infection, we characterize the protective efficacy and immune responses of mice intramuscularly injected with plasmid encoding DEN-2 non-structural protein 1 (NS1). Intravenously challenged by lethal DEN-2, mice vaccinated with NS1-DNA exhibited a delay onset of paralysis, a marked decrease of morbidity, and a significant enhancement of survival. In addition to a moderate increase of NS1-specific antibody titer from immunized mice measured by ELISA, a strong priming effect on anti-NS1 response was also noticed in plasmid NS1-vaccinated mice by radioimmunoprecipitation (RIP) or immunoblot analysis. Interestingly, newborn mice from NS1-DNA-immunized dam showed stronger resistance to viral challenge, as compared to those from vector DNA or PBS-immunized dams, indicating the protective role of NS1-specific antibody. In contrast to humoral immune response, DNA immunization can elicit strong cellular immune responses, including NS1-specific T cell proliferation and cytolytic activity. The NS1-DNA-induced protection can be further augmented by co-injection of plasmid encoding interleukin 12 (IL-12), suggesting an effector role of Th1 immunity against DEN infection. In summary, our results suggest the potential of NS1-DNA vaccine against DEN infection, and indicate both NS1-specific humoral and cellular immune responses contribute to the protection.

Immunomodulatory effect of decoy receptor 3 on the differentiation and function of bone marrow-derived dendritic cells in nonobese diabetic mice: from regulatory mechanism to clinical implication.

To investigate the regulatory effects of decoy receptor 3 (DcR3) on the differentiation and function of dendritic cells (DCs), bone marrow‐derived DCs (BM‐DCs) from nonobese diabetic (NOD) mice were cultured with recombinant DcR3.Fc protein. Their differentiating phenotypes and T cell‐stimulating functions were then evaluated. Expression of CD11c, CD40, CD54, and major histocompatibility complex I‐Ag7 was reduced in cells cultured with additional DcR3.Fc, compared with DCs incubated with granulocyte macrophage‐colony stimulating factor and interleukin (IL)‐4, indicating that DcR3 interferes with the differentiation and maturation of BM‐DCs. One of the most striking effects of DcR3.Fc on the differentiation of DCs was the up‐regulation of CD86 and down‐regulation of CD80, suggesting a modulatory potential to skew the T cell response toward the T helper cell type 2 (Th2) phenotype. Consistent with this, the proliferation of CD4+ T cells cocultured with DcR3.Fc‐treated DCs was significantly reduced compared with that of T cells stimulated by normal DCs. Moreover, the secretion of interferon‐γ from T cells cocultured with DcR3.Fc‐treated DCs was profoundly suppressed, indicating that DcR3 exerts a Th1‐suppressing effect on differentiating DCs. Furthermore, adoptive transfer experiments revealed that NOD/severe combined immunodeficiency mice received DcR3.Fc‐treated DCs, and subsequently, autoreactive T cells showed delayed onset of diabetes and a decrease in diabetic severity compared with mice that received normal DCs and T cells, suggesting a future therapeutic potential in autoimmune diabetes. Data from two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization‐time‐of‐flight analysis show an up‐regulation of some proteins—such as mitogen‐activated protein kinase p38 β, cyclin‐dependent kinase 6, and signal‐induced proliferation‐associated gene 1—and a down‐regulation of the IL‐17 precursor; tumor necrosis factor‐related apoptosis‐inducing ligand family member‐associated nuclear factor‐κB activator‐binding kinase 1; and Golgi S‐nitroso‐N‐acetylpenicillamine in cells treated with DcR3, further demonstrating its effect on DC differentiation and function.

Water quality parameters associated with prevalence of Legionella in hot spring facility water bodies.

Some species of Legionella are recognized as opportunistic potential human pathogens, such as Legionella pneumophila, which causes legionnaires disease. Indeed, outbreaks of legionellosis are frequently reported in areas in which the organism has been spread via aerosols from contaminated institutional water systems. Contamination in hot tubs, spas and public baths are also possible. As a result, in this study, we investigated the distribution of Legionella at six hot spring recreation areas throughout Taiwan. Legionella were detected in all six hot spring recreation areas, as well as in 20 of the 72 samples that were collected (27.8%). Seven species of Legionella identified from samples by the direct DNA extraction method were unidentified Legionella spp., Legionella anisa, L. pneumophila, Legionella erythra, Legionella lytica, Legionella gresilensis and Legionella rubrilucen. Three species of Legionella identified in the samples using the culture method were L. pneumophila, unidentified Legionella spp. and L. erythra. Legionella species were found in water with temperatures ranging from 22.7 °C to 48.6 °C. The optimal pH appeared to range from 5.0 to 8.0. Taken together, the results of this survey confirmed the ubiquity of Legionella in Taiwan spring recreational areas. Therefore, a long-term investigation of the health of workers at hot spring recreational areas and the occurrence of Legionella in hot spring recreational areas throughout Taiwan are needed.

Differentiation and identification of Shigella spp. and enteroinvasive Escherichia coli in environmental waters by a molecular method and biochemical test.

Both Shigella spp. and enteroinvasive Escherichia coli (EIEC) are important human pathogens that are responsible for the majority of cases of endemic bacillary dysentery. However, they are difficult to identify and differentiate by biochemical tests or molecular methods alone. In this study, we developed a procedure to detect Shigella spp. and EIEC from environmental water samples using membrane filtration followed by nutrient broth enrichment, isolation using selective culture plates, and identification of the invasion plasmid antigen H (ipaH) gene by PCR amplification and DNA sequencing. Finally, we used a biochemical test and a serological assay to differentiate between Shigella and EIEC. Among the 93 water samples from nine reservoirs and one watershed, 76 (81.7%) water samples of culture plates had candidate colonies of Shigella and EIEC and 5 water samples were positive (5.4%) for a Shigella- and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the molecular method, the biochemical test, and the serological assay, 11 ipaH gene-positive isolates from 5 water samples were all identified as EIEC.

Low-Dose 5-Aza-2'-deoxycytidine Pretreatment Inhibits Experimental Autoimmune Encephalomyelitis by Induction of Regulatory T Cells

Forkhead box P3 (Foxp3) is the major transcription factor controlling the development and function of regulatory T (Treg) cells. Previous studies have indicated epigenetic regulation of Foxp3 expression. Here, we investigated whether the deoxyribonucleic acid (DNA) methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza) applied peripherally could modulate central nervous system (CNS) inflammation, by using a mouse experimental autoimmune encephalomyelitis (EAE) model. We found that disease activity was inhibited in a myelin oligodendrocyte glycoprotein (MOG) peptide-induced EAE mouse briefly pretreated with low-dose (0.15 mg/kg) 5-Aza, ameliorating significant CNS inflammatory responses, as indicated by greatly decreased proinflammatory cytokines. On the contrary, control EAE mice expressed high levels of IFN-γ and interleukin (IL)-17. In addition, 5-Aza treatment in vitro increased GFP expression in CD4+GFP− T cells isolated from GFP knock-in Foxp3 transgenic mice. Importantly, 5-Aza treatment increased Treg cell numbers, in EAE mice, at both disease onset and peak. However, Treg inhibition assays showed 5-Aza treatment did not enhance per-cell Treg inhibitory function, but did maintain a lower activation threshold for effector cells in EAE mice. In conclusion, 5-Aza treatment prevented EAE development and suppressed CNS inflammation, by increasing the number of Treg cells and inhibiting effector cells in the periphery.

SOX2 modulates alternative splicing in transitional cell carcinoma

Aberrant alternative splicing of key cellular regulators may play a pivotal role in cancer development. To investigate the potential influence of altered alternative splicing on the development of transitional cell carcinoma (TCC), splicing activity in the TCC cell lines TSGH8301 and BFTC905 was examined using the SV40-immortalized uroepithelial cell line SV-HUC-1 as a reference. Our results indicate a significant alteration in splice site selection in the TCC cell lines. By gene expression profiling and subsequent validation, we discovered that sex-determining region Y-box protein 2 (SOX2) is specifically upregulated in BFTC905. Furthermore, ectopic expression of SOX2 modulates alternative splicing of the splicing reporter in vivo. More significantly, using an in vitro pull-down assay, it was found that SOX2 exhibits RNA-binding capability. Our observations suggest that SOX2 modulates alternative splicing by functioning as a splicing factor.

Methylomics analysis identifies ZNF671 as an epigenetically repressed novel tumor suppressor and a potential non-invasive biomarker for the detection of urothelial carcinoma.

The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved. Here, by methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in bladder UC tumor tissue samples, a finding that was independently validated by bisulphite pyrosequencing in cell lines and tissue samples. Subsequent assays including treatment with epigenetic depressive agents and in vitro methylation showed ZNF671 methylation to result in its transcriptional repression. ZNF671 re-expression in UC cell lines, via ectopic expression, inhibited tumor growth and invasion, in possible conjunction with downregulation of cancer stem cell markers (c-KIT, NANOG, OCT4). Clinically, high ZNF671 methylation in UC tumor tissues (n=96; 63 bladder, 33 upper urinary tract) associated with tumor grade and poor locoregional disease-free survival. Quantitative MSP analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical decision-making.

Identification of a nuclear localization signal in the polo box domain of Plk1

Polo-like kinase 1 plays an essential role in mitosis and cytokinesis. Expression and nuclear localization of Plk1 during the S phase are necessary for its functions. Although it was reported that a bipartite nuclear localization signal located at the N-terminal kinase domain is required for nuclear import of Plk1, Plk1 carrying mutations in the polo box I of the polo box domain exhibited increased cytoplasmic accumulation. We further showed that the polo box domain was able to confer nuclear import of beta-galactosidase in vivo and GST-EGFP in vitro. The import carriers transportin and importin alpha were found to interact with the polo box domain directly in a Ran-GTP sensitive manner. These results indicate the presence of a nuclear localization signal in the polo box domain. A 38 amino acid sequence with the function of nuclear localization signal was identified to interact with transportin. Our findings demonstrated that a transportin-dependent nuclear localization signal is present in the polo box domain of Plk1, possibly required for efficient nuclear import. Showing little similarity to the M9 sequence, the 38 amino acid sequence identified here likely represents a novel nuclear localization signal.

Elevated frequency and function of regulatory T cells in patients with active chronic hepatitis C

BackgroundRegulatory T cells (Tregs) play a pivotal role in the persistence of hepatitis C virus infection. The aim of this study was to evaluate the frequency and function of Tregs in patients with chronic hepatitis C (CHC).MethodsWe enrolled 44 CHC patients with elevated alanine aminotransferase (ALT) levels (CH group), 13 CHC patients with persistent normal ALT levels (PNALT group), and 14 age-matched healthy subjects (HS group; controls). Tregs were identified as CD4+, CD25+, and forkhead box P3 (Foxp3)+ T lymphocytes, using three-color fluorescence-activated cell sorting (FACS). The frequency of Tregs was determined by calculating the percentage of CD4+CD25high T cells among CD4 T cells. CD127 and CD45RA were also analyzed for subsets of Tregs. The levels of serum transforming growth factor (TGF)-β and interleukin (IL)-10 in immunosuppressive assays were detected by enzyme-linked immunosorbent assay (ELISA). The immunosuppressive abilities of Tregs were evaluated by measuring their ability to inhibit the proliferation of effector cells.ResultsHigher proportions of Tregs were found in the CH and PNALT groups compared with the HS group. The populations of CD127 low/negative and CD45RA negative cells were higher in the CH group than in the PNALT group. The expressions of IL-10 and TGF-β in the CH and PNALT groups were significantly higher than those in the HS group. In addition, the immunosuppressive ability of Tregs from the CH group was increased relative to that in the PNALT and the HS group.ConclusionsCHC patients, irrespective of liver function, had higher frequencies of Tregs than healthy subjects; however, only CHC patients with inflammation showed enhanced immunosuppressive function of Tregs.