The modulation of hypoxia-inducible factor-1α/plasminogen activator inhibitor-1 axis in human gingival fibroblasts stimulated with cyclosporine A

Background/PurposeThe prominent side effect of the immunosuppressive drug cyclosporine A (CsA) is gingival overgrowth. Hypoxia-inducible factor (HIF)-1α regulates a wide variety of profibrogenic genes, which are closely associated with tissue fibrosis. The aim of this study was to compare HIF-1α expression in normal gingival tissues and CsA-induced gingival overgrowth specimens and further explore the potential mechanisms that may lead to induction of HIF-1α expression.MethodsFifteen CsA-induced gingival overgrowth specimens and five normal gingival tissues were examined by immunohistochemistry. Western blot was used to investigate the effects of CsA on the expression of HIF-1α in cultured human gingival fibroblasts. The effects of CsA on plasminogen activator inhibitor (PAI)-1 expression were evaluated in environmental hypoxia.ResultsHIF-1α staining in gingival tissue was stronger in CsA-induced gingival overgrowth group than normal gingival group (p < 0.05). The expression of HIF-1α was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p = 0.041). CsA was found to upregulate HIF-1α protein in a dose-dependent manner (p < 0.05). Hypoxia increased CsA-induced PAI-1 protein expression than normoxic conditions (p < 0.05).ConclusionThese results suggest that HIF-1α expression is significantly upregulated in CsA-induced gingival overgrowth specimens. The activation of HIF-1α may promote fibrogenesis by an increase of PAI-1 expression and a subsequent elevation of extracellular matrix production in gingival tissues

A Novel Single-Dose Dengue Subunit Vaccine Induces Memory Immune Responses

To protect against dengue viral infection, a novel lipidated dengue subunit vaccine was rationally designed to contain the consensus amino acid sequences derived from four serotypes of dengue viruses. We found that the lipidated consensus dengue virus envelope protein domain III (LcED III) is capable of activating antigen-presenting cells and enhancing cellular and humoral immune responses. A single-dose of LcED III immunization in mice without extra adjuvant formulation is sufficient to elicit neutralizing antibodies against all four serotypes of dengue viruses. In addition, strong memory responses were elicited in mice immunized with a single-dose of LcED III. Quick, anamnestic neutralizing antibody responses to a live dengue virus challenge were elicited at week 28 post-immunization. These results demonstrate the promising possibility of a future successful tetravalent vaccine against dengue viral infections that utilizes one-dose vaccination with LcED III

Male Germ Cell-Specific RNA Binding Protein RBMY: A New Oncogene Explaining Male Predominance in Liver Cancer

Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen-peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome-specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC

Dengue-1 Envelope Protein Domain III along with PELC and CpG Oligodeoxynucleotides Synergistically Enhances Immune Responses

The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus

Molecular Characterization of Ahp2, a Lytic Bacteriophage of

is an opportunistic pathogen that infects fish, amphibians, mammals, and humans. This study isolated a myophage, vB_AhyM_Ahp2 (Ahp2), that lytically infects . We observed that 96% of the Ahp2 particles adsorbed to within 18 min. Ahp2 also showed a latent period of 15 min with a burst size of 142 PFU/cell. This phage has a linear double-stranded DNA genome of 47,331 bp with a GC content of 57%. At least 20 Ahp2 proteins were detected by SDS-polyacrylamide gel electrophoresis; among them, a 40-kDa protein was predicted as the major capsid protein. Sequence analysis showed that Ahp2 has a genome organization closely related to a group of phages (13AhydR10RR, 14AhydR10RR, 85AhydR10RR, phage 3, 32 Asp37, 59.1), which infect and . The tail module encompassing ORF27-29 in the Ahp2 genome was present in all phages analyzed in this study and likely determines the host range of the virus. This study found that Ahp2 completely lyses AH300206 in 3.5 h at a MOI of 0.0001 and does not lysogenize its host. Altogether, these findings show that Ahp2 is a lytic phage and could be a candidate for therapeutic phage cocktails

Bisgma stimulates prostaglandin E2 production in macrophages via cyclooxygenase-2, cytosolic phospholipase A2, and mitogen-activated protein kinases family.

BACKGROUND: Bisphenol A-glycidyl-methacrylate (BisGMA) employs as a monomer in dental resins. The leakage of BisGMA from composite resins into the peripheral environment can result in inflammation via macrophage activation. Prostaglandin E2 (PGE2) is a key regulator of immunopathology in inflammatory reactions. Little is known about the mechanisms of BisGMA-induced PGE2 expression in macrophage. The aim of this study was to evaluate the signal transduction pathways of BisGMA-induced PGE2 production in murine RAW264.7 macrophages. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we demonstrate that BisGMA can exhibit cytotoxicity to RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). In addition, PGE2 production, COX-2 expression, and cPLA2 phosphorylation were induced by BisGMA on RAW264.7 macrophages in a dose- and time-dependent manner (p<0.05). Moreover, BisGMA could induce the phosphorylation of ERK1/2 pathway (MEK1/2, ERK1/2, and Elk), p38 pathway (MEK3/6, p38, and MAPKAPK2), and JNK pathway (MEK4, JNK, and c-Jun) in a dose- and time-dependent manner (p<0.05). Pretreatment with AACOCF3, U0126, SB203580, and SP600125 significantly diminished the phosphorylation of cPLA2, ERK1/2, p38, and JNK stimulated by BisGMA, respectively (p<0.05). BisGMA-induced cytotoxicity, cPLA2 phosphorylation, PGE2 generation, and caspases activation were reduced by AACOCF3, U0126, SB203580, and SP600125, respectively (p<0.05). CONCLUSIONS: These results suggest that BisGMA induced-PGE2 production may be via COX-2 expression, cPLA2 phosphorylation, and the phosphorylation of MAPK family. Cytotoxicity mediated by BisGMA may be due to caspases activation through the phosphorylation of cPLA2 and MAPKs family

Upregulation of Slug expression by cyclosporine A contributes to the pathogenesis of gingival overgrowth

Gingival overgrowth occurs as a side effect of systemic medication with immunosuppressant cyclosporine A (CsA). Slug, a master regulator of epithelial–mesenchymal transition, is dramatically upregulated in a variety of fibrotic diseases. The aim of this study is to investigate the role of epithelial–mesenchymal transition marker Slug in the pathogenesis of CsA-induced gingival overgrowth. Methods: Clinically healthy gingiva and CsA-induced gingival overgrowth specimens were analyzed by immunohistochemistry. The effect of CsA on normal human gingival fibroblasts (HGFs) was used to elucidate whether Slug expression could be affected by CsA by real-time reverse transcription-polymerase chain reaction and western blot. Cell proliferation in CsA-treated HGFs with Slug lentiviral-mediated shRNAi knockdown was evaluated by tetrazolium bromide reduction assay. Results: Slug expression was higher in CsA-induced gingival overgrowth specimens than in clinical healthy gingiva (p < 0.05). Slug expression was significantly higher in CsA-induced gingival overgrowth specimens with higher levels of inflammatory infiltrates (p < 0.05). CsA was found to increase Slug transcript and protein expression in HGFs in a dose-dependent manner (p < 0.05). In addition, knockdown of Slug significantly suppressed CsA-induced cell proliferation in HGFs (p < 0.05). Conclusion: Taken together, upregulation of Slug in HGFs stimulated by CsA may play an important role in the pathogenesis of CsA-induced gingival overgrowth

Stable Isotope-Labeled Lipidomics to Unravel the Heterogeneous Development Lipotoxicity

Non-alcoholic fatty liver disease (NAFLD) as a global health problem has clinical manifestations ranging from simple non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis, and cancer. The role of different types of fatty acids in driving the early progression of NAFL to NASH is not understood. Lipid overload causing lipotoxicity and inflammation has been considered as an essential pathogenic factor. To correlate the lipid profiles with cellular lipotoxicity, we utilized palmitic acid (C16:0)- and especially unprecedented palmitoleic acid (C16:1)-induced lipid overload HepG2 cell models coupled with lipidomic technology involving labeling with stable isotopes. C16:0 induced inflammation and cell death, whereas C16:1 induced significant lipid droplet accumulation. Moreover, inhibition of de novo sphingolipid synthesis by myriocin (Myr) aggravated C16:0 induced lipoapoptosis. Lipid profiles are different in C16:0 and C16:1-treated cells. Stable isotope-labeled lipidomics elucidates the roles of specific fatty acids that affect lipid metabolism and cause lipotoxicity or lipid droplet formation. It indicates that not only saturation or monounsaturation of fatty acids plays a role in hepatic lipotoxicity but also Myr inhibition exasperates lipoapoptosis through ceramide in-direct pathway. Using the techniques presented in this study, we can potentially investigate the mechanism of lipid metabolism and the heterogeneous development of NAFLD

Effects of BisGMA on the phosphorylation of p38 pathway.

<p>(A) RAW264.7 macrophages were stimulated with 3 µM BisGMA for the indicated treatment periods. Cells were harvested and protein extracts were subjected to SDS-PAGE. Western blot analysis using antibodies against the phosphorylated and total p38. (B) Cells were stimulated with the indicated concentrations of BisGMA for 15 min. (C) After treatment with 30 µM SB203580 for 30 min, cells were stimulated with 3 µM BisGMA for 15 min. Cells were harvested and protein extracts were subjected to SDS-PAGE Western blot analysis using antibodies against phosphorylated and total MEK3/6, p38, and MAPKAPK-2. The ratio of immunointensity between the phosphorylation and total protein was calculated. Values are expressed as mean ± SD (n = 3). *represents significant difference from control values with <i>p</i><0.05. #represents significant difference from BisGMA values with <i>p</i><0.05.</p