Response to immune complex vaccine in chronic hepatitis B patients is associated with lower baseline level of serum IgG galactosylation.

The composition of glycan in immunoglobulin G (IgG) has shown to affect various diseases and can be regulated by drugs and preventive vaccination. A hepatitis B surface antigen (HBsAg)-hepatitis B immunoglobulin (HBIG) immune complex (YIC) therapeutic vaccine for chronic hepatitis B (CHB) patients has undergone clinical trials. To explore for markers of CHB, which could be associated with responsiveness to YIC therapeutic vaccine, serum IgG glycosylation in CHB patients was analyzed.Kinetic changes of serum galactosylated IgG in 53 hepatitis Be antigen (HBeAg)-positive CHB patients treated with YIC were monitored by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) analysis. Whole blood cytokines were assayed by cytokine binding assay kits. All samples were back assayed before treatment, during therapy and follow-up for 6 months from a previous completed clinical trial.During YIC treatment, 26 patients with lower IgG galactosylation level at baseline [galactosylation level (Gal-ratio) = -0.29, 0.18 (mean, SD)] showed sustained increase of serum galactosylated IgG, and responded to YIC treatment by HBeAg seroconversion. While those who did not respond to YIC treatment [Gal-ratio = -0.40, 0.15 (mean, SD)] failed to show similar changes. Furthermore, this kinetic increase of galactosylated IgG correlated with marked up-regulated IL-2 level, confirming that effective cellular immune responses have participated in responsiveness.For HBeAg-positive CHB patients lower serum IgG galactosylation level may serve as an indicator for selecting a suitable subpopulation of candidates for YIC therapeutic vaccination.

Decreased expression of hepatocyte nuclear factor 4α (Hnf4α)/microRNA-122 (miR-122) axis in hepatitis B virus-associated hepatocellular carcinoma enhances potential oncogenic GALNT10 protein activity.

MicroRNA-122 (miR-122), a mammalian liver-specific miRNA, has been reported to play crucial roles in the control of diverse aspects of hepatic function and dysfunction, including viral infection and hepatocarcinogenesis. In this study, we explored the clinical significance, transcriptional regulation, and direct target of miR-122 in hepatitis B virus (HBV)-associated hepatocellular carcinoma. Reduced expression of miR-122 in patients with HBV-associated hepatocellular carcinoma was correlated with venous invasion and poor prognosis. Furthermore, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-10 (GALNT10) was identified as a bona fide target of miR-122 in hepatoma cells. Ectopic expression and knockdown studies showed that GALNT10 indeed promotes proliferation and apoptosis resistance of hepatoma cells in a glycosyltransferase-dependent manner. Critically, adverse correlation between miR-122 and GALNT10, a poor prognosticator of clinical outcome, was demonstrated in hepatoma patients. Hepatocyte nuclear factor 4α (Hnf4α), a liver-enriched transcription factor that activates miR-122 gene transcription, was suppressed in HBV-infected hepatoma cells. Chromatin immunoprecipitation assay showed significantly reduced association of Hnf4α with the miR-122 promoter in HBV-infected hepatoma cells. Moreover, GALNT10 was found to intensify O-glycosylation following signal activation of the epidermal growth factor receptor. In addition, in a therapeutic perspective, we proved that GALNT10 silencing increases sensitivity to sorafenib and doxorubicin challenge. In summary, our results reveal a novel Hnf4α/miR-122/GALNT10 regulatory pathway that facilitates EGF miR-122 activation and hepatoma growth in HBV-associated hepatocarcinogenesis.

Estrogen receptor α (ERα) mediates 17ß-estradiol (E2)-activated expression of HBO1.

BACKGROUND: HBO1 (histone acetyltransferase binding to ORC1) is a histone acetyltransferase (HAT) which could exert oncogenic function in breast cancer. However, the biological role and underlying mechanism of HBO1 in breast cancer remains largely unknown. In the current study, we aimed to investigate the role of HBO1 in breast cancer and uncover the underlying molecular mechanism. METHODS: Immunohistochemistry was applied to detect HBO1 protein expression in breast cancer specimens (n=112). The expression of protein level was scored by integral optical density (IOD) for further statistical analyses using SPSS. Real-time PCR was used to simultaneously measure mRNA levels of HBO1. The HBO1 protein expression in breast cancer cells was confirmed by western blot. RESULTS: HBO1 was highly expressed in breast cancer tissues and significantly correlated with estrogen receptor α (ERα) (p<0.001) and progestational hormone (PR) (p=0.002). HBO1 protein level also correlated positively with histology grade in ERα positive tumors (p=0.016) rather than ERα negative tumors. 17ß-estradiol (E2) could upregulate HBO1 gene expression which was significantly inhibited by ICI 182,780 or ERα RNAi. E2-increased HBO1 protein expression was significantly suppressed by treatment with inhibitor of MEK1/2 (U0126) in T47 D and MCF-7 cells. CONCLUSIONS: HBO1 was an important downstream molecule of ERα, and ERK1/2 signaling pathway may involved in the expression of HBO1 increased by E2.

Cyclin G associated kinase interacts with interleukin 12 receptor beta2 and suppresses interleukin 12 induced IFN-gamma production.

Interleukin 12 receptor beta1 (IL-12Rbeta1) and beta2 (IL-12Rbeta2) constitute the functional and high-affinity receptor complex for interleukin 12 (IL-12) and mediate important functions in activated T cells. In this study, we identified cyclin G associated kinase (GAK) as a new IL-12Rbeta2-interacting protein using yeast two-hybrid system and confirmed it by coimmunoprecipitation assays. Overexpression of GAK in activated T cells suppresses IL-12 induced IFN-gamma production but has no detectable effects on its proliferation, whereas knockdown of GAK by RNA interference (RNAi) increases IFN-gamma production. These results suggest that GAK associates with IL-12Rbeta2 and may play a role in regulating IL-12 signaling.

Specific immune response to HBsAg is enhanced by beta-glucan oligosaccharide containing an alpha-(1-->3)-linked bond and biased towards M2/Th2.

Vaccination with Hepatitis B surface antigen (HBsAg) is being used to prevent HBV infection. The fact that 10% of vaccinees fail to develop protective antibodies has fostered a large body of research for more effective vaccination strategies. Search for new adjuvant, able to selectively trigger protective antibody production, is one of the most promising approaches. The oligosaccharide beta-(1-->6)-branched beta-(1-->3) glucohexaose is the basic unit of lentinan and several other fungal beta-glucans with immunostimulatory activity. beta-glucans stimulate innate immune response mainly through interaction with myeloid cells (macrophages) and dendritic cells. In this study, the ability of synthetic beta-(1-->6)-branched beta-(1-->3) glucohexaose analogue (beta-glu6) to enhance the immune response to HBsAg has been evaluated. Administration of synthetic beta-glu6 i.p. in BALB/c mice greatly enhanced the mobilisation and maturation of macrophages and dendritic cells to co-administered HBsAg, as compared to the antigen alone. The adjuvant effect of beta-glu6 was evident in the increase of T and B cell activation in response to HBsAg, as judged by the percentage of CD69-positive CD4(+) and CD19(+) lymphocytes in the spleen. beta-glu6 could significantly enhance the number of IL-4-producing cells in response to HBsAg, while it had no effect on the number of IFN-gamma-producing lymphocytes, suggesting a Th2 bias of the immune response. The correlate of protection for HBV vaccination, i.e. the titer of HBsAg-specific antibodies, was greatly enhanced by the use of beta-glu6 as a vaccine adjuvant. The IgG1/IgG2a ratio within the anti-HBsAg antibodies was higher in the mice immunised with HBsAg plus beta-glu6 than receiving HBsAg alone or mice administered HBsAg with Freund's adjuvant, indicating a shift towards a Th2-biased anti-inflammatory protective antibody response.

Studies on the beta(l-4)Galactosyltransferase of Cell Surface in Induced HL60 Cells.

A fluorescene assay method for beta(1-4) galactosyltransferase (beta1-4GT) of cell surface has been developed using a pyridylaminated sugar as an acceptor substrate. A fluorescent sugar chain, whose reducing end of the Gnbeta1-2Malpha1-6(Gnbeta1-2Malpha1-3) Mbeta1-4Gnbeta1-4(Fucalpha1-6) Gn has been aminated with 2-aminopyridine. beta1-4GT activity of cell surface varied in different stages of the cell cycle with the highest activity at interphase. The enzyme activity of cell surface took a change when HL60 cell line was induced to differentiate by PMA or RA. The cell surface enzyme activity was 1.30 times as much as tile control group at 24 h in the case of PMA induction, and a maximum increase of 70% over the control on the third day with RA induction.

Regulatory Effect of All-trans Retinoic Acid (ATRA) on the Activities of N-acetylglucosaminyl Transferase (GnT) III, IV in HL-60 Cells.

The regulatory effect of all-trans retinoic acid (ATRA) on the activities of N-acetylglucosaminyl transferase (GnT) III and IV in HL-60 Cells were studied. It was found that the activities of GnT-III and GnT-IV were significantly decreased by 0.1 &mgr;M ATRA, but not further decreased with the increase in concentration of ATRA to 1.0 or 10 &mgr;M. The activities of both GnT-III and GnT-IV in the untreated control cells were variable during the incubation of cells, showing a peak level at 24 h and 48 h in the un-synchronized culture and the synchronized culture respectively. After the treatment with ATRA, the peak value was still at 24h in un-synchronized culture, and the activities of GnT-III and GnT-IV were gradually decreased only after 24 h of incubation both in un- synchronized and synchronized culture of HL-60 cells. The above results were discussed.

Studies on the Method for the Determination of beta1-4 Galactosyltransferase and Its Kinetics.

A fluorescence assay method for beta1-4galactosyltransferase (beta1-4GT) has been developed involving a pyridylaminated sugar as an acceptor substrate, a fluorescent sugar chain, with the reducing end of the Gnbeta1 - 2Malpha1 - 6(Gnbeta1-2Malpha1-3)Mbeta1 - 4Gnbeta1 - 4Gn - PA aminated with 2-aminopyridine. Microsome was prepared from the liver of normal male rats as an enzyme sample. Then the fluorescent reaction product was separated by reverse-phase HPLC. The kinetic experiments were carried out using crude enzyme extracts of the Golgi complex from the rat liver. The enzyme has a pH optimum of 6.5,and optimal concentration of Triton X-100 of 0.5%. The K(m) and V(max) values for the sugar acceptor substrates were found to be 2.31x10(-3)M(-1) and 5.75x10(-2) &mgr;mol/(min.mg) respectively. Furthermore, our research revealed that beta1-4GT had branch specificity. The Gn of alpha1-3 mannose branch of the acceptor substrate was more susceptible to galactosylation than that of the alpha1-6 branch by 2.10 times.

Changes in Glycosylation Alter the Affinity of the Human Transferrin for Its Placental Receptor.

By using serial lectin-Sepharose affinity chromatography (ConA, DSA, and LCA), the transferrins carrying the biantennary complex type of oligosaccharides from sera of healthy individuals and those carrying multiantennary oligosaccharides from sera of pregnant women were purified. The two kinds of transferrins were used to study their binding affinity for isolated placental transferring receptor. The results showed that the binding affinity of the highly branched transferring decreased to half of that for the biantennary type with a K(d) of 9.80x10(-8) M as against the latter's 4.97x10(-8) M while the maximum binding remains constant. These results suggested that changes in glycosylation altered the affinity of the human transferrin for its placental receptor.

The regulation of N-Acetylglucosaminyl Transferase III by Protein Kinase C.

The activity of N-acetylglucosaminyl transferase III (GnT III) in 7721 human heptocarcinoma cell was inhibited by two non-specific Ser/Thr protein kinase inhibitors, quercetin and trifluoperazine, and two PKC specific inhibitors, D-sphingosine and staurosporine. The change of GnT III activity paralleled that of membranous PKC (m-PKC) when the cells were treated with PMA, but not with that of cytosolic PKC (c-PKC). Quercetin, D-sphingosine and staurosporine also blocked the PMA activation of GnT-III. The inhibitory effects on m-PKC and GnT III were generally proportional to the concentration of quercetin and staurosporine. When crude GnT preparations of human and rat kidney were treated with ALP to remove the phosphate groups, the GnT III activities were significantly decreased. These results suggest that GnT III may be regulated by m-PKC directly or indirectly via the phosphorylation/dephosphorylation of the Ser/Thr residue.