Vitamin C acts indirectly to modulate isotype switching in mouse B cells / 대한해부학회지

Vitamin C, one of essential micronutrients, has been reported to modulate the humoral immune responses in some mammals. We investigated whether vitamin C might modulate this response in mice by directly affecting B cells. Splenic B cells were isolated and activated by CD40- and B cell receptor-ligation in vitro. The cells were cultured with a pretreatment of vitamin C from 0 to 1 mM of concentrations. Vitamin C slightly increased apoptosis of B cells dose-dependently and behaved as an antioxidant. We found that in vivo administration of vitamin C by intraperitoneal injection affected isotype switching as previously reported: the titer of antigen-specific IgG1 antibody was decreased, while that of IgG2a was unaffected. Somewhat different from those observed in vivo, in vitro exposure to vitamin C slightly decreased isotype switching to IgG1 and increased isotype switching to IgG2a. Pretreatment with vitamin C in the safe range did not affect either proliferation of cultured B cells or the expression of CD80 and CD86 in those cells. Taken together, in vivo results suggest that vitamin C acts to modulate isotype switching in the mouse. However, because of our in vitro results, we suggest that the modulation exerted by vitamin C in vivo is by indirectly affecting B cells, perhaps by directly influencing other immune cells such as dendritic cells.

Mega-dose vitamin C attenuated lung inflammation in mouse asthma model / 대한해부학회지

Asthma is a Th2-dependent disease mediated by IgE and Th2 cytokines, and asthmatic patients suffer from oxidative stresses from abnormal airway inflammation. Vitamin C is a micro-nutrient functioning as an antioxidant. When administered at a mega-dose, vitamin C has been reported to shift immune responses toward Th1. Thus, we tried to determine whether vitamin C exerted beneficial effects in asthma animal model. Asthma was induced in mice by sensitizing and challenging with ovalbumin. At the time of challenge, 3~5 mg of vitamin C was administered and the effects were evaluated. Vitamin C did not modulate Th1/Th2 balance in asthma model. However, it decreased airway hyperreactivity to methacholine, decreased inflammatory cell numbers in brochoalveolar lavage fluid, and moderate reduction of perivascular and peribronchiolar inflammatory cell infiltration. These results suggest that vitamin C administered at the time of antigen challenge exerted anti-inflammatory effects. Further studies based on chronic asthma model are needed to evaluate a long-term effect of vitamin C in asthma. In conclusion, even though vitamin C did not show any Th1/Th2 shifting effects in this experiment, it still exerted moderate anti-inflammatory effects. Considering other beneficial effects and inexpensiveness of vitamin C, mega-dose usage of vitamin C could be a potential supplementary modality for the management of asthma.

N-acetyl-L-Cysteine Inhibits Functional Activation of Mouse Bone Marrow-Derived Dendritic Cells / 대한해부학회지

N-acetyl-L-cysteine (NAC) is a thiol-containing compound and acts as a precursor for glutathione (GSH). It behaves as an antioxidant in mammalian cells and also exerts anti-inflammatory effects. NAC is also known to affect several immune cells including eosinophils, B cells, T cells, and dendritic cells (DC) in many aspects. Even though it has been reported that NAC inhibits DC activation and shifts the immune response to Th2, these studies exhibit some contradictory results in detail and do not give any information with respect to the induction of regulatory T cells. In this study, we re-analyzed the effects of NAC on DC during their activation. We also evaluated whether it induced T cell anergy, Th1/Th2 shift, or regulatory T cells. NAC suppressed the elevation of intracellular reactive oxygen species during DC activation. In parallel, it down-regulated surface expression of CD40 and CD86, suppressed the decrease of phagocytic function, lowered the secretion of cytokines such as IL-6, IL-10, and IL-12. All these effects showed dose-dependency. Thus, it seems likely that NAC inhibited DC activation with regard to their phenotype and cytokine secretion. When we evaluated the T cell-stimulating capacity of these NAC-DC, T cell proliferation and secretion of both Th1 (IFN-gamma) and Th2 cytokine (IL-5) were decreased. This implies that the T cell-stimulating activity of NAC-DC decreased without any shift to Th1 or Th2 cytokine (IL-5). The secretion of IL-10 and TGF-beta in the supernatants were also decreased, which suggests that the decrease of T cell proliferation and cytokine secretion is due to the induction of T cell anergy, rather than regulatory T cells.

Development of Human Antibody Inhibiting RNase H Activity of Polymerase of Hepatitis B Virus Using Phage Display Technique

BACKGROUND: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. METHODS: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. RESULTS: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of 4.46 X 10(9) cfu was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of 4.5 X 10(-7) M and 1.9 X 10(-7) M, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. CONCLUSION: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.