Chien-Hui Hung

Inhibition of Helicobacter pylori-induced inflammation in human gastric epithelial AGS cells by Phyllanthus urinaria extracts

AIM OF THE STUDY Helicobacter pylori is linked to a majority of peptic ulcers and to some types of gastric cancer, and its resistance to antibiotic treatment is now found worldwide. This study is aimed at evaluating the antimicrobial activity of Phyllanthus urinaria Linnea (Euphorbiaceae), chloroform (PUC) and methanol (PUM) extracts, and its eight isolates on H. pylori-infected human gastric epithelial AGS cells. MATERIALS AND METHODS The in vitro anti-bacterial activity of P. urinaria chloroform (PUC) and methanol (PUM) extracts, and its eight isolates were determined. Additional experiments were also performed to know the PUC and PUM ability to inhibit the H. pylori adhesion to and invasion of AGS cells, in addition to the effect of PUC on NF-kappaB activity as well as IL-8 synthesis during H. pylori infection of AGS cells. RESULTS The results revealed that crude extracts PUC and PUM showed potent antimicrobial activity against H. pylori than pure isolates. On the other hand, in vitroH. pylori-infection model revealed that the inhibition of bacterial adhesion and invasion to AGS cells has dramatically reduced by treatment of extract PUC, while PUM has the same moderate effect. Furthermore, H. pylori-induced nuclear factor (NF)-kappaB activation, and the subsequent release of interleukin (IL)-8 in AGS cells were also inhibited by the extract PUC. CONCLUSIONS These results open the possibility of considering P. urinaria a chemopreventive agent for peptic ulcer or gastric cancer, but this bioactivity should be confirmed in vivo in the future.

Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway

ABSTRACT Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of Epstein-Barr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development. IMPORTANCE Autophagy is a cellular process that degrades and recycles nutrients under stress conditions to promote cell survival. Although autophagy commonly serves as a defense mechanism against viral infection, many viruses exploit this mechanism to promote their replication. This study finds that a transcription factor that is encoded by Epstein-Barr virus (EBV), Rta, activates autophagy, and the inhibition of autophagy reduces the ability of the virus to express viral lytic proteins and to generate progeny. Unlike other virus-encoded proteins that modulate autophagy by interacting with proteins that are involved in the autophagic pathway, Rta activates the transcription of the autophagy-related genes via the ERK pathway. The results of this study reveal how the virus manipulates autophagy to promote its lytic development.

Characterization and intracellular trafficking of Epstein-Barr virus BBLF1, a protein involved in virion maturation

ABSTRACT Epstein-Barr virus (EBV) BBLF1 shares 13 to 15% amino acid sequence identities with the herpes simplex virus 1 UL11 and cytomegalovirus UL99 tegument proteins, which are involved in the final envelopment during viral maturation. This study demonstrates that BBLF1 is a myristoylated and palmitoylated protein, as are UL11 and UL99. Myristoylation of BBLF1 both facilitates its membrane anchoring and stabilizes it. BBLF1 is shown to localize to the trans-Golgi network (TGN) along with gp350/220, a site where final envelopment of EBV particles takes place. The localization of BBLF1 at the TGN requires myristoylation and two acidic clusters, which interact with PACS-1, a cytosolic protein, to mediate retrograde transport from the endosomes to the TGN. Knockdown of the expression of BBLF1 during EBV lytic replication reduces the production of virus particles, demonstrating the requirement of BBLF1 to achieve optimal production of virus particles. BBLF1 is hypothesized to facilitate the budding of tegumented capsid into glycoprotein-embedded membrane during viral maturation.

A/C polymorphism in the interleukin-18 coding region among Taiwanese systemic lupus erythematosus patients:

Interleukin-18 (IL-18) is associated with chronic inflammation, autoimmune diseases and various cancers and infectious diseases. An IL-18 genetic A/C polymorphism at coding position 105 (rs549908) has been linked with asthma and rheumatoid arthritis. We tested a hypothesis that the IL-18 genetic polymorphism confers systemic lupus erythematosus (SLE) susceptibility. Study participants were Taiwanese SLE patients and a healthy control group. Our results indicate (1) a significantly higher A allele frequency in SLE patients (P = 0.003; OR = 1.97; 95% CI = 1.26—3.08) and (2) a significantly higher A allele frequency in SLE patients with a central nervous system disorder (P = 0.027; OR = 7.18; 95% CI = 0.95—54.28). Our results suggest that the A/C polymorphism contributes to SLE pathogenesis. Lupus (2008) 17, 124—127.

Role of the cellular transcription factor YY1 in the latent-lytic switch of Kaposi's sarcoma-associated herpesvirus.

Lytic cycle reactivation of Kaposis sarcoma-associated herpesvirus (KSHV) is initiated by expression of the ORF50 gene. Here we show that YY1 protein specifically binds to the ORF50 promoter (ORF50p) region in vitro and in vivo. After treatment with chemical inducers, including sodium butyrate (SB) and TPA, the levels of YY1 protein are inversely correlated with the lytic induction of KSHV in cells. Overexpression of YY1 completely blocks the ORF50p activation in transient reporter assays, while mutation at the YY1 site in the ORF50p or knockdown of YY1 protein confers an enhancement of the ORF50p activation induced by SB and TPA. YY1 overexpression in a stable cell clone HH-B2(Dox-YY1) also inhibits expression of the ORF50 and its downstream lytic genes. On the other hand, a chimeric YY1 construct that links to its coactivator E1A can disrupt viral latency. These results imply that YY1 is involved in the regulation of KSHV reactivation.

ORF50-dependent and ORF50-independent activation of the ORF45 gene of Kaposi's sarcoma-associated herpesvirus

The ORF45 gene of Kaposis sarcoma-associated herpesvirus (KSHV) encodes a multifunctional tegument protein. Here, we characterize the transcriptional control of the ORF45 gene and show that its promoter can be activated by ORF50 protein, a latent-lytic switch transactivator. The ORF45 promoter can also be induced by sodium butyrate (SB), a histone deacetylase inhibitor, in the absence of ORF50 protein. Although SB induces the ORF45 gene independently of ORF50, its full activation may require the presence of ORF50. Deletion and point mutation analyses revealed that two RBP-Jκ-binding sites in the ORF45 promoter confer the ORF50 responsiveness, whereas NF-Y and Sp1-binding sites mediate the response to SB. Direct binding of NF-Y, Sp1, or RBP-Jκ protein to the ORF45 promoter is required for the promoter activation induced by SB or by ORF50. In conclusion, our study demonstrates both ORF50-dependent and ORF50-independent transcriptional mechanisms operated on the activation of the ORF45 gene.

Positive and negative regulation in the promoter of the ORF46 gene of Kaposi's sarcoma-associated herpesvirus.

The ORF46 gene of Kaposis sarcoma-associated herpesvirus (KSHV) encodes uracil DNA glycosylase, an enzyme involved in DNA repair. In this study, we show that the transcriptional start site of the ORF46 gene is located at nucleotide 69,425 of the viral genome and ORF50 protein, a latent-lytic switch transactivator, activates the ORF46 promoter via RBP-Jκ protein. Three consensus RBP-Jκ-binding sites found in the ORF46 promoter are critical for the binding of RBP-Jκ protein and conferring the ORF50 responsiveness. In addition, a negative regulatory region has been determined in the ORF46 promoter, which mediates the suppression of the ORF50 responsiveness. The functional negative region of the ORF46 promoter is mainly composed of the Sp1-binding sites. Like the negative region of the ORF46 promoter, addition of Sp1-binding sequences alone in an ORF50-responsive promoter efficiently confers the suppression of the ORF50 responsiveness. Furthermore, sodium butyrate, a pleiotropic inducing agent for the KSHV lytic cycle, is able to relieve the negative regulation of the ORF46 promoter in the latently KSHV-infected cells. The identification of multiple positive and negative cis-acting regulatory elements in the viral promoters emphasizes the elaborate controls in the KSHV lytic cycle, which ensure the adequate expression of each viral lytic gene.

Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus

ABSTRACT The orf47-orf46-orf45 gene cluster of Kaposis sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle. IMPORTANCE Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.

Young children with Behçet disease have more intestinal involvement.

Objective: Gastrointestinal (GI) involvement in childhood Behçet disease (BD) is not well understood. We aimed to clarify the intestinal presentation in children with BD. Methods: Medical records of 85 children with recurrent oral ulcers between 1990 and 2010 at the National Taiwan University Hospital were reviewed retrospectively. Twenty of them who fulfilled the Mason and Barnes criteria for the diagnosis of childhood BD were enrolled. The clinical and laboratory characteristics were analyzed. Results: Among 20 patients, the median age at diagnosis was 13.2 years. The common presentations included oral ulcers (100%), genital ulcers (70%), skin lesions (65%), and GI symptoms (50%). Five of 10 patients with GI symptoms received endoscopic examinations and all had ulcers. Divided by the age of 10, patients younger than 10 years tended to have higher rates of GI symptoms initially and intestinal ulcers (P = 0.002 and 0.015, respectively). Platelet count was significantly lower in young patients (P = 0.0151). Patients without GI symptoms had higher rates of skin involvement than patients with GI symptoms (P = 0.019). Conclusions: Young children with BD tended to have more GI presentations. For children with BD younger than 10 years having GI symptoms, endoscopic examinations may be considered.

Cellular senescence occurring in the rabbit medial collateral ligament during healing.

Medial collateral ligament (MCL) healing proceeds in a temporally ordered fashion after injury. Despite the critical roles of fibroblasts during ligament repair, the phenotypic features of these healing fibroblasts have not been well characterized. Here, we show that healing MCL fibroblasts obtained from rabbits at 3‐week postinjury exhibited higher rates of senescent phenotypes and produced higher levels of TGF‐β1, collagens, α‐SMA, and matrix metalloproteinases (MMPs), than the corresponding fibroblasts from sham‐operated MCLs. Mechanical stretch further enhanced the cellular senescence and the expression of TGF‐β1, collagens, α‐SMA, and MMPs in both sham and healing MCL fibroblasts. In addition to MCL fibroblasts at 3‐week postinjury, the increased cellular senescence was also detected in healing MCL fibroblasts obtained at 4‐ and 6‐week postinjury. Most importantly, the association between the cellular senescence and ligament healing was confirmed in tissue sections by the senescence‐associated β‐galactosidase (SA‐β‐gal) staining. Using a recombinant TGF‐β1 and a neutralizing antibody, we found that those phenotypic changes, such as cellular senescence and the expression of collagens and MMPs, in MCL fibroblasts under mechanical loading conditions were regulated through TGF‐β1. Taken together, our results propose that cellular senescence and turnover of extracellular matrixes regulated by TGF‐β1 in MCL fibroblasts are critical for ligament healing.