[Relationship between the suppressor of cytokine signaling 3 expression and antiviral efficacy of nucleos(t)ide and interferon alpha therapy for chronic hepatitis B].

Objective: To investigate the molecular mechanism of poor response of nucleoside and interferon therapy in some patients with chronic hepatitis B (CHB) and the negative regulatory factor of suppressor of cytokine signaling 3 (SOCS3) expression in the interferon-signaling pathway. Also, study the clinical relationship between SOCS3 and antiviral efficacy of nucleoside and interferon. Methods: Peripheral blood and matched liver tissue samples from 54 CHB patients who participated in the OSST study were selected. HBsAg was measured at different time points (baseline and weeks 12, 24, 36, and 48) to observe the antiviral efficacy. Meanwhile, quantitative real-time PCR, and immunohistochemistry were used to detect the expression levels of SOCS3 mRNA in peripheral blood mononuclear cells (PBMCs) and matched liver tissues (baseline and 48 weeks). At the end of the 48-week treatment, patients with HBsAg negative or HBeAg seroconversion were defined as response group, and vice versa. Paired t-tests were used to compare normal distribution variables and the Mann-Whitney U test was used to compare the median differences between groups of non-normally distributed variables. Results: After 48 weeks of treatment, serum HBsAg levels in the Peg-IFN group continued to decline (average decrease of 1.14 log(10) IU / ml at week 48; P = 0.001 compared with baseline), while the entecavir group remained almost unchanged during treatment (average decrease was 0.05 log(10) IU / ml at week 48; compared with baseline P = 0.12). The expression of SOCS3 mRNA (Messenger RNA, mRNA) in peripheral blood and liver tissues of non-responder group was significantly higher than the response group in the course of Peg-IFNα2a treatment. The immunohistochemical results of liver tissue showed that the expression of SOCS3 in the non-responder group was significantly higher than that in the response group at baseline (P = 0.027). After 48 weeks of treatment with Peg-IFNα2a, the expression of SOCS3 in the non-responder group was significantly higher than that in the baseline and response groups (P = 0.003, P = 0.012, respectively). Conclusion: The expression of SOCS3 in peripheral blood mononuclear cells and liver tissues of non-responding CHB patients was significantly higher than that of responding CHB patients during interferon and nucleoside antiviral therapy. We speculated that SOCS3 might affect the antiviral efficacy through negative regulation of JAK-STAT signaling pathway, and partly expose the mechanism of interferon resistance.

[Liposome nanoparticles for targeted delivery of parthenolide induce colorectal cancer necroptosis to ameliorate tumor-infiltrating T cell exhaustion in mice].

OBJECTIVE: To investigate the mechanism of parthenolide for inducing necroptosis and ameliorating CD8+ T cell exhaustion in colorectal cancer (CRC) and construct liposome nanoparticles for targeted delivery of parthenolide. METHODS: The inhibitory effect of parthenolide on proliferation of different CRC cell lines was examined using CCK8 assay, and ROS LDH detection and Western blotting were used to analyze the cell death pathways. In a mouse model bearing subcutaneous MC38 cell xenografts, the effects of 5 and 15 mg/kg parthenolide on tumor growth and CD8+ T cell depletion were observed. In a mouse model bearing orthotopic CRC cell xenograft in the ileocecal region, free parthenolide (100 µg/mL) or low (100 µg/mL) and high doses (200 µg/mL) of liposome nanoparticles loaded with parthenolide were injected via the tail vein, and the changes in CD8 expression in the xenografts were analyzed using immunohistochemistry. RESULTS: Treatment with parthenolide dose-dependently lowered the viability of the CRC cell lines SW480, DLD1, HCT116 and MC38 cells, and its effect was obviously antagonized by Nec-1. Immunoblotting analysis showed that parthenolide treatment resulted in increased RIP3 and MLKL phosphorylation in the CRC cells. In the mouse model bearing subcutaneous xenografts, parthenolide treatment at the high dose, but not at the low dose, significantly increased the number of infiltrating CD3+ CD8+ T cells and PD1hiTIM3+ T cell percentage (P<0.01) and lowered the percentage of PD1loTIM3- T cells in the tumor tissue (P<0.01). In the mouse models bearing orthotopic CRC xenograft, intravenous injection of the liposomes loaded with parthenolide, especially at the high dose, significantly increased CD8 expression in the tumor tissue (P<0.01). CONCLUSION: Parthenolide induces necroptosis in CRC and increases infiltrating CD8+ T cells to ameliorate CD8+ T cell exhaustion in the tumor. Liposome nanoparticles for targeted delivery of parthenolide produce stronger, anti-tumor effect.

Effectiveness of egg yolk immunoglobulin (IgY) against periodontal disease-causing Fusobacterium nucleatum.

AIMS: To evaluate the in vitro and in vivo effectiveness of egg yolk immunoglobulin (IgY) against periodontal disease-causing Fusobacterium nucleatum. METHODS AND RESULTS: Four White Leghorn hens (120 days old) were immunized with whole Fus. nucleatum cells killed with 1% formaldehyde using three injections provided at 2-week intervals. IgY was produced from egg yolks obtained from these immunized hens using water dilution, two-step salt precipitation and ultrafiltration. This IgY was shown to have a purity of 86·8% based on its optical intensity in the stained SDS-PAGE bands. An enzyme-linked immunosorbent assay indicated a high specificity for the IgY against Fus. nucleatum with a maximum antibody titre of 80 000. The IgY had only weak cross-reactivity with Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei. Growth and biofilm formation by Fus. nucleatum were inhibited by IgY at concentrations of 10 and 20 mg ml(-1) . Immunofluorescence and immunoelectron microscope assays revealed a high binding ability of specific IgY, which may explain the in vitro effectiveness of IgY. In an in vivo study, IgY treatment resulted in a marked decrease in alveolar bone loss after Fus. nucleatum infection in a mouse model confirming the effectiveness of IgY against periodontal disease-causing Fus. nucleatum. CONCLUSIONS: IgY effectively inhibited growth and biofilm formation by Fus. nucleatum and prevented the progression of periodontal disease by decreasing alveolar bone loss. SIGNIFICANCE AND IMPACT OF THE STUDY: Specific IgY may have potential for the treatment of periodontal disease.

Mitigating Biases in a Cohort Study: A Critical Examination of the Silicone Arthroplasty in Rheumatoid Arthritis Study.

Research bias, or the systematic errors of a study, can arise before, during, or after a trial ends. These biases hinder the internal validity of the study, which is the accuracy of a study's conclusions regarding the effects of an intervention on a given group of subjects. With the growing use of evidence-based medicine, there is a demand for high-quality evidence from the research community. Systematic reviews and meta-analyses of randomized controlled trials are considered the highest level of evidence, followed by individual randomized controlled trials. However, most surgical trials cannot be conducted as randomized controlled trials because of factors such as patient preferences and lack of equipoise among surgeons. Therefore, surgical trials may lack features that are held as important standards for high-quality evidence, such as randomization and blinding. To demonstrate the biases that surgical trials may encounter, the authors examined a prospective cohort study, the Silicone Arthroplasty in Rheumatoid Arthritis study. The authors focus on the challenges that arise during a surgical trial, including the design, implementation, and methods used to report the clinical evidence. By recognizing and addressing obstacles that exist in research, investigators will provide health care providers with high-quality evidence needed to make well-informed, evidence-based clinical decisions.

Construct for high-level expression and low misincorporation of lysine for arginine during expression of pET-encoded eukaryotic proteins in Escherichia coli.

The arginine codon AGA is rarely used in E. coli but is common in eukaryotic genes. Prior studies have shown that the low level of tRNA(UCUArg) can lead to low expression and misincorporation of lysine for arginine, during expression of genes containing AGA codons in E. coli. The chloramphenicol-selectable plasmid pJY2 is designed to facilitate the expression of such genes cloned into pET vectors: it encodes T7 lysozyme (to depress constitutive expression of the cloned gene) and tRNA(UCUArg) (to suppress lysine misincorporation at AGA codons). Using pJY2, we observed robust and translationally faithful expression of mutant ubiquitin genes in which 14% (11 out of 76) of the total codons were AGA. Competent BL21(DE3)pJY2 cells can be used to suppress lysine misincorporation and achieve high-level expression of pET-encoded target genes without modification of AGA codons in the target gene sequence.

A novel pH-sensitive membrane from chitosan--TEOS IPN; preparation and its drug permeation characteristics.

A novel organic-inorganic composite membrane was prepared, using tetra ethyl ortho silicate (TEOS) as an inorganic material and chitosan as an organic compound. Equilibrium and oscillatory swelling studies were conducted to investigate swelling behaviors of the membrane according to the pH of the swelling medium. Drug permeation experiments were also performed in phosphate buffer solution of the pH of 2.5 and 7.5, respectively. Lidocaine HCl, sodium salicylate and 4-acetamidophenol were selected as model drugs to examine the effect of ionic property of drug on the permeation behavior. The effects of membrane composition and the external pH on the swelling and the drug permeation behavior of IPN membrane could be summarized as follows; chitosan incorporated into TEOS IPN swelled at pH 2.5 while shrunk at pH 7.5. This swelling behavior was completely reversible and the membrane responded rapidly to the change in environmental pH condition. According to swelling behavior, an increase in pH from 2.5 to 7.5 yielded an increase in the rate of drug permeation because of the shrinking of the incorporated chitosan in TEOS IPN, while decrease in pH resulted in low permeation rate. The optimal TEOS-chitosan ratio for maximum pH-sensitivity existed and drug permeation was influenced not only with the external pH but also with the ionic interactions between the drug and membrane.

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.

Kuwanon G: an antibacterial agent from the root bark of Morus alba against oral pathogens.

Kuwanon G was isolated from the ethyl acetate fraction of methanol extract of Morus alba and its structure was elucidated by 13C-NMR, 1H-NMR and FAB-MS. Antibacterial activity of kuwanon G was investigated by the minimum inhibitory concentration (MIC) test and the viable cell count method. MIC of kuwanon G against Streptococcus mutans causing dental caries was determined to be 8.0 microg/ml. The bactericidal test showed that kuwanon G completely inactivated S. mutans at the concentration 20 microg/ml in 1 min. Kuwanon G also significantly inhibited the growth of other cariogenic bacteria such as Streptococcus sobrinus and Streptococcus sanguis, and Porpyromonas gingivalis causing periodontitis. Transmission electron microscopy (TEM) of kuwanon G treated cells demonstrated remarkable morphological damage of the cell wall and condensation of the cytoplasm.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

A HECT domain E3 enzyme assembles novel polyubiquitin chains.

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.

E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains.

Polyubiquitin (Ub) chains linked through Lys-48-Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K(m) of approximately 9 microM for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.

Calorimetric characterization of the formation of acrylic type bone cements.

The formation of acrylic bone cements upon heating was investigated by differential scanning calorimetry (DSC). The effects of the contents of initiators, accelerator, biocompatibilizer, and crosslinking agents on the rate and the heat of polymerization during DSC heating were studied. The rate and the heat of polymerization (delta H) were characterized by the peak temperature and the area of the DSC exotherm, respectively. It was found that both the rate and heat of polymerization decreased with increasing heating rate. The delta H was increased considerably with increasing benzoyl peroxide (BPO) initiator concentration from 1 to 10% (w/v), whereas the rate of polymerization was reduced significantly. An increase in azobisisobutyronitrile (AIBN) initiator concentration also induced an increase in delta H, but the rate of reaction was not affected considerably. The addition of accelerator promoted the rate of reaction but resulted in a drop in delta H. The rate of polymerization for the system containing BPO initiator was increased quite significantly with the addition of hydroxyethyl methacrylate (HEMA) biocompatibilizer, while the delta H was slightly increased. For the system using AIBN as the initiator, the rate of polymerization was decreased slightly and the delta H dropped significantly with the addition of HEMA. The effect of ethylene glycol dimethacrylate (EGDMA) crosslinking agent was also examined. Polymerization became more rapid with the addition of EGDMA in the bone cement using BPO initiator, while it remained approximately constant for the system using AIBN as the initiator. No systematic change in delta H was observed with the addition of EGDMA in both systems. This study demonstrated that DSC is a potential tool to measure the amount of heat released and also the rate of polymerization for bone cements.

Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.

TRAF6 is a signal transducer in the NF-kappaB pathway that activates IkappaB kinase (IKK) in response to proinflammatory cytokines. We have purified a heterodimeric protein complex that links TRAF6 to IKK activation. Peptide mass fingerprinting analysis reveals that this complex is composed of the ubiquitin conjugating enzyme Ubc13 and the Ubc-like protein Uev1A. We find that TRAF6, a RING domain protein, functions together with Ubc13/Uev1A to catalyze the synthesis of unique polyubiquitin chains linked through lysine-63 (K63) of ubiquitin. Blockade of this polyubiquitin chain synthesis, but not inhibition of the proteasome, prevents the activation of IKK by TRAF6. These results unveil a new regulatory function for ubiquitin, in which IKK is activated through the assembly of K63-linked polyubiquitin chains.

Helicobacter pylori in dental plaque and saliva.

BACKGROUND: About half of the world population is infected with H. pylori, but the transmission and the source of this infection are still unclear. Recently, dental plaque (DP) and saliva have been implicated as possible sources of H. pylori infection. This study was done to investigate the detection rates of H. pylori in the DP and saliva by use of PCR depending on H. pylori infection state of gastric mucosa. METHODS: In 46 subjects, gastric H. pylori colonization was evaluated with CLO test, microscopy of Gram stained mucosal smear, culture and histology after modified Giemsa staining in the antrum and body, respectively. A patient was regarded as H. pylori positive if one or more of the four aforementioned test methods demonstrated H. pylori colonization of the gastric mucosa. For detection of H. pylori in the DP and saliva, PCR assay was done with ET4-U and ET4-L primers. To estimate the sensitivity and specificity of this PCR, H. pylori positivity was evaluated in the antrum and body, separately. RESULTS: The sensitivity of mucosal PCR was 50.0% (27/54) and the specificity 86.8% (33/38). When a subject was regarded as H. pyloi positive, if either antrum or body mucosal H. pylori was is positive, the positive rate of mucosal PCR was 62.1% (18 subjects) in the 29 H. pylori-positive and 17.6% (3 subjects) in the 17 H. pylori-negative subjects. DP PCR was positive in 2 of 29 H. pylori-positive subjects (6.9%) and none in the 17 H. pylori-negative (0%). Saliva PCR was positive in 4 of 14 H. pylori-positive subjects (28.6%) and none of 6 H. pylori-negative (0%). CONCLUSION: The detection rates of H. pylori in DP and saliva by PCR were rather low, 6.9% and 28.6%, respectively, and these rates might have been underestimated by low sensitivity of the PCR method used in this study. However, the results that H. pylori was found in the DP and saliva suggest that the oral cavity can perform a role as a reservoir of H. pylori in Korea.

Intraperitoneal gene delivery mediated by a novel cationic liposome in a peritoneal disseminated ovarian cancer model.

We have previously synthesized a new cationic liposome that displays high efficiency and low toxicity, 3 beta[l-ornithinamide-carbamoyl] cholesterol (O-Chol), using solid-phase synthesis. In this study, O-Chol was applied to in vitro and in vivo models of ovarian cancer. Intraperitoneal gene delivery for peritoneal disseminated ovarian cancer in nude mice was achieved using a stable chloramphenicol acetyl transferase (CAT)-expressing ovarian cancer cell line (OV-CA-2774/CAT), which allowed us to quantify the exact tumor burden of organs. When luciferase and beta-galactosidase genes were used as reporter genes, O-Chol showed better efficiency than other commercial transfection reagents such as lipofectin, lipofectAMINE, DC-Chol, and FuGENE 6, both in vitro and in vivo. Moreover, the transfection efficiency of this new cationic lipid reagent remained high in serum-containing medium and under serum-free conditions. Furthermore, in vivo transfection with O-Chol showed high levels of gene expression specific to peritoneal tumor cells. Consequently, the O-Chol:DNA lipoplex appears to offer potential advantages over other commercial transfection reagents because of (1) its higher level of gene expression in vitro and in vivo; (2) its reduced susceptibility to serum inhibition; and (3) its highly selective transfection into tumor cells. These results suggest that the O-Chol:DNA lipoplex is a promising tool in gene therapy for patients with peritoneal disseminated ovarian cancer.

The pre-styloid compartment of the parapharyngeal space: a three-dimensional digitized model based on the Chinese Visible Human.

To build a digitized visible model of the parapharyngeal space of the Chinese Visible Human and to provide a sectional anatomic basis for radiological and clinical diagnosis of the parapharyngeal space, sectional anatomy data of the parapharyngeal space were selected from the Chinese Visible Human male and female to compare with MR imaging findings in the axial planes. From these data the parapharyngeal space and surrounding structures were segmented. They were then reconstructed in three dimensions on PC. In the axial planes of the sectional anatomy and MR imaging, the shape, content and relations of the parapharyngeal space were clearly displayed and the dominant plane for showing the parapharyngeal space was elicited. The three-dimensional reconstructed images displayed perfectly the anatomic relationships of the parapharyngeal space, parotid, muscles, mandible and vessels. All reconstructed structures can be displayed singly, in groups or as a whole; any diameter or angle of the reconstructed structures can be easily measured. The Chinese Visible Human male and female data set can provide complete and accurate data. The digitized model of the parapharyngeal space and its surroundings offers unique insights into the complex anatomy of the area, providing morphologic data for imaging diagnosis and surgery of the parapharyngeal space.