Suhkneung Pyo

Immunostimulating effects of acidic polysaccharides extract of Panax ginseng on macrophage function.

ABSTRACT The root of Panax ginseng C. A. Meyer is one of the most popular natural tonics in oriental countries. In this study, we have isolated polysaccharide fraction of Panax ginseng (ginsan) and examined its effect on the function of murine peritoneal macrophages. When macrophages were treated with ginsan, cytotoxic activity against B16 melanoma cells was significantly induced. In addition, the levels of cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and lnterferon-γ (IFN-γ) were increased and the production of reactive oxygen/nitrogen components such as nitric oxide (NO) and hydrogen peroxide (H2O2) was enhanced. Moreover, phagocytic activity was induced in ginsan-treated macrophages compared to the control. The expression of CD14 and l-Ab on murine peritoneal macrophages was increased by the treatment with ginsan, while the expression of CD11b was decreased. Taken together, these results suggest that ginsan has an immunopotentiating effects on macrophages and these abilities could be used clinically for the treatment of diseases such as cancer.

Reversal of P-glycoprotein-mediated multidrug resistance by ginsenoside Rg3

Multidrug resistance has been a major problem in cancer chemotherapy. In this study, in vitro and in vivo modulations of MDR by ginsenoside Rg(3), a red ginseng saponin, were investigated. In flow cytometric analysis using rhodamine 123 as an artificial substrate, Rg(3) promoted accumulation of rhodamine 123 in drug-resistant KBV20C cells in a dose-dependent manner, but it had no effect on parental KB cells. Additionally Rg(3) inhibited [3H]vinblastine efflux and reversed MDR to doxorubicin, COL, VCR, and VP-16 in KBV20C cells. Reverse transcriptase-polymerase chain reaction and immuno-blot analysis after exposure of KBV20C cells to Rg(3) showed that inhibition of drug efflux by Rg(3) was due to neither repression of MDR1 gene expression nor Pgp level. Photo-affinity labeling study with [3H]azidopine, however, revealed that Rg(3) competed with [3H]azidopine for binding to the Pgp demonstrating that Rg(3) competed with anticancer drug for binding to Pgp thereby blocking drug efflux. Furthermore, Rg(3) increased life span in mice implanted with DOX-resistant murine leukemia P388 cells in vivo and inhibited body weight increase significantly.

Induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan.

The immunomodulatory effect of ginsan based on the production of cytokines and the activation of macrophage was studied. Murine peritoneal macrophages (PM) on in vitro treatment with ginsan isolated from Panax ginseng induced mRNA of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin-1 (IL-1)beta, interleukin-6 (IL-6) and interleukin-12 (IL-12); TNF-alpha mRNA induction was maximum within 3 h, IL-6 mRNA was gradually induced up to 24 h, and IL-1beta and IL-12 mRNA were highly induced at 24 h. IL-1beta and IL-6 protein levels also increased within 24 h in a dose-dependent manner and reached a maximum with 100 microg/ml ginsan. IL-12 was induced after 3 days and a high level of induction was detected after 4 days post treatment. Ginsan enhanced the lytic death of L929 cells through TNF-alpha activation. The mRNA expression of nitric oxide synthase (iNOS) was highly induced after 24 h treatment of ginsan, and then NO production was maximum after 48-h treatment with a low dose of 1 microg/ml. The level of iNOS mRNA induction by ginsan was slightly less than that of macrophages activating agents such as LPS plus IFN-gamma. The tumoricidal activity of macrophage cultured with ginsan on Yac-1 cells was enhanced in a dose-dependent manner; growth inhibition increased 1.6-fold with 100 microg/ml ginsan. These results suggest that ginsan exerts as an effective immunomodulator and enhances antitumor activity of macrophages.

Effect of Heat Shock and Mutations in ClpL and ClpP on Virulence Gene Expression in Streptococcus pneumoniae

ABSTRACT Spread of Streptococcus pneumoniae from the nasopharynx to other host tissues would require the organism to adapt to a variety of environmental conditions. Since heat shock proteins are induced by environmental stresses, we investigated the effect of heat shock on ClpL and ClpP synthesis and the effect of clpL and clpP mutations on the expression of key pneumococcal virulence genes. Pulse labeling with [35S]methionine and chase experiments as well as immunoblot analysis demonstrated that ClpL, DnaK, and GroEL were stable. Purified recombinant ClpL refolded urea-denatured rhodanese in a dose-dependent manner, demonstrating ClpLs chaperone activity. Although growth of the clpL mutant was not affected at 30 or 37°C, growth of the clpP mutant was severely affected at these temperatures. However, both clpL and clpP mutants were sensitive to 43°C. Although it was further induced by heat shock, the level of expression of ClpL in the clpP mutant was high at 30°C, suggesting that ClpP represses expression of ClpL. Furthermore, the clpP mutation significantly attenuated the virulence of S. pneumoniae in a murine intraperitoneal infection model, whereas the clpL mutation did not. Interestingly, immunoblot and real-time reverse transcription-PCR analysis demonstrated that pneumolysin and pneumococcal surface antigen A were induced by heat shock in wild-type S. pneumoniae. Other virulence genes were also affected by heat shock and clpL and clpP mutations. Virulence gene expression seems to be modulated not only by heat shock but also by the ClpL and ClpP proteases.

The ClpP Protease of Streptococcus pneumoniae Modulates Virulence Gene Expression and Protects against Fatal Pneumococcal Challenge

ABSTRACT Streptococcus pneumoniae usually colonizes the nasopharynx of humans asymptomatically but occasionally translocates from this niche to the lungs, the brain, and the blood, causing potentially fatal infections. Spread to other host tissues requires a significant morphological change and the expression of virulence factors, such as capsular polysaccharide, and virulence proteins, such as pneumolysin (Ply), PspA, and CbpA. Modulation of the expression of pneumococcal virulence genes by heat shock and by heat shock proteins ClpL and ClpP, as well as the attenuation of virulence of a clpP mutant in a murine intraperitoneal infection model, was demonstrated previously. In this study, we further investigated the underlying mechanism of virulence attenuation by the clpP mutation. The half-lives of the mRNAs of ply and of the first gene of the serotype 2 capsule synthesis locus [cps(2)A] in the clpP mutant were more than twofold longer than those of the parent after heat shock, suggesting that the mRNA species were regulated posttranscriptionally by ClpP. In addition, the clpP mutant was defective in colonization of the nasopharynx and survival in the lungs of mice after intranasal challenge. The mutant was also killed faster than the parent in the murine macrophage RAW264.7 cell line, indicating that ClpP is required for colonization and intracellular survival in the host. Furthermore, fractionation studies demonstrated that ClpP was translocated into the cell wall after heat shock, and immunization of mice with ClpP elicited a protective immune response against fatal systemic challenge with S. pneumoniae D39, making ClpP a potential vaccine candidate for pneumococcal disease.

West Nile virus capsid protein induces p53‐mediated apoptosis via the sequestration of HDM2 to the nucleolus

The capsid protein of the West Nile virus (WNV) functions as an apoptotic agonist via the induction of mitochondrial dysfunction and the activation of caspases‐9 and ‐3. Here, we have determined that the WNV capsid (WNVCp) is capable of binding to and sequestering HDM2 into the nucleolus. WNVCp was shown to interfere with the formation of the HDM2 and p53 complex, thereby causing the stabilization of p53 and the subsequent induction of its target apoptotic protein, Bax. Whereas WNVCp was capable of inducing the p53‐dependent apoptotic process in wild‐type mouse embryonic fibroblasts (MEF) or SH‐SY5Y cells, it exerted no significant effects on p53‐null MEF or on p53‐knockdown SH‐SY5Y cells. This suggests that WNVCp‐mediated apoptosis requires p53. Furthermore, when WNV was transfected into cells, endogenous Hdm2 and WNVCp were able to interact physically. WNVCp expressed in wild‐type MEF proved able to induce the translocation of the endogenous Hdm2 into the nucleolus. Consistently, WNV was highly pathogenic in the presence of p53, and was less so in the absence of p53. The results of these studies suggest that the apoptotic mechanism mediated by WNV might occur in accordance in a fashion similar to that of the tumour‐suppressing mechanism mediated by ARF.

Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein.

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G2 phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.

Diosgenin inhibits macrophage-derived inflammatory mediators through downregulation of CK2, JNK, NF-κB and AP-1 activation

Diosgenin is a precursor of steroid hormones, which can be found in several plant species. Diosgenin has been shown to have a variety of biological activities including anti-inflammatory activity, but through a mechanism that is unclear. Especially, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of diosgenin on the production of inflammatory mediators in lipopolysaccharide (LPS)/interferon gamma (IFN-gamma)-activated murine macrophage. Macrophages pre-exposed to diosgenin (0.1-10 microM) were stimulated with LPS/IFN-gamma. Pretreatment with diosgenin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In addition, diosgenin inhibits production of reactive oxygen species (ROS), interleukin-1 (IL-1), and IL-6, but not that of tumor necrosis factor-alpha (TNF-alpha). Inhibition of these inflammatory mediators appears to be at the transcriptional level, since diosgenin decreased LPS/IFN-gamma-induced NF-kappaB and AP-1 activity. Diosgenin blocked CK2 activation and phosphorylation of c-Jun NH(2)-terminal kinase (JNK), but not that of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of inflammatory mediators in macrophages. Taken together the present data suggest that diosgenin reduces the production of inflammatory meditators by inhibiting LPS/IFN-gamma-triggered CK2, JNK, NF-kappaB and AP-1 activation, thereby implicating a mechanism by which diosgenin may exert its immunosuppressive effects.

Inhibition of TNF-α-induced adhesion molecule expression by diosgenin in mouse vascular smooth muscle cells via downregulation of the MAPK, Akt and NF-κB signaling pathways.

Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-α in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2h with diosgenin (0.1-10 μM) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-α induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-κB activation induced by TNF-α. Furthermore, diosgenin inhibited TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NF-κB. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-α-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-κB signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.

Methamphetamine administration produces immunomodulation in mice.

The abuse of methamphetamine (MA) is an increasingly growing problem globally and produces serious side effects. In the present study, the immunomodulating effects of MA were examined on the immune system after MA (5 mg/kg body weight) was administered daily orally for 14 d. The immune system was evaluated by the antibody response to sheep red blood cells (SRBC; plaque assay and serum immunoglobulin [Ig] G), natural killer (NK) activity, lymphocyte subpopulations in the spleen and thymus, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. Body weight, spleen weight, and thymus weight generally decreased in MA-treated mice. MA treatment induced an increase in the percentage of CD4+ cells with simultaneous decrease in the percentages of CD8+ and double-positive CD4+CD8+ in thymus. MA inhibited the IgM plaque-forming cell number, and lowered the level of IgG, the proliferation of mitogen-stimulated B and T cells, and the growth of granulocyte–macrophage colony-forming units (CFU-GM). Exposure to MA also decreased interleukin-2 production by splenocytes. In contrast, splenic NK activity in exposed mice was significantly enhanced. Taken together, data indicate that the immune system was suppressed by oral MA exposure.