Protective properties of geniposide against UV-B-induced photooxidative stress in human dermal fibroblasts.

CONTEXT: Geniposide (genipin-1-O-ß-d-glucoside) is a major bioactive ingredient in the fruits of gardenia [Gardenia jasminoides J. Ellis (Rubiaceae)], a traditional herbal medicine in Asian countries. OBJECTIVE: This work assesses the skin anti-photoaging potential of geniposide in human dermal fibroblasts under UV-B irradiation. MATERIALS AND METHODS: The anti-photoaging property of geniposide, at varying concentrations (5, 12 and 30 µM) treated for 30 min prior to UV-B irradiation, was evaluated by analysing reactive oxygen species (ROS), promatrix metalloproteinase-2 (proMMP-2), glutathione (GSH), superoxide dismutase (SOD), nuclear factor erythroid 2-related factor 2 (Nrf2) and cellular viability. RESULTS: Geniposide suppressed the ROS elevation under UV-B irradiation, which was revealed using three ROS-sensitive fluorescent dyes. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE) and dihydrorhodamine 123 (DHR-123) elicited the IC50 values of 10.5, 9.8 and 21.0 µM, respectively. Geniposide attenuated proMMP-2 at activity and protein levels that were elevated under UV-B-irradiation. Geniposide at 5, 12 and 30 µM augmented the UV-B-reduced total GSH content to 1.9 ± 0.1-, 2.2 ± 0.2- and 4.1 ± 0.2-fold, respectively. Geniposide at 5, 12 and 30 µM upregulated total SOD activity to 2.3 ± 0.1-, 2.5 ± 0.3- and 3.3 ± 0.3-fold, respectively, under UV-B irradiation. The UV-B-reduced Nrf2 levels were also upregulated by geniposide treatment. Geniposide, at the concentrations used, was unable to interfere with cellular viabilities under UV-B irradiation. DISCUSSION AND CONCLUSIONS: After the skin anti-photoaging potential of geniposide may be further verified, it can be utilized as a safer resource in the manufacture of effective anti-aging cosmetics.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production.

PURPOSE: The goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies. MATERIALS AND METHODS: DNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells. RESULTS: Twelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone. CONCLUSION: DNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Tumor regression is mediated via the induction of HER263-71- specific CD8+ CTL activity in a 4T1.2/HER2 tumor model: no involvement of CD80 in tumor control.

In the CT26/HER2 and 4T1.2/HER2 tumor models, CT26/HER2 cells form tumors that continue to grow, whereas 4T1.2/HER2 cells form tumors that eventually regress. Here, we investigated the differences in the behaviors of these two cell lines. When immune cells from 4T1.2/HER2 tumor-bearing animals were stimulated with HER2 class I peptides, they displayed a 2-fold increase in IFN-γ levels, in response to the peptides, HER263-71 and HER2342-350. In contrast, extremely high levels of antigen-non-specific IFN-γ production were observed with immune cells and sera from CT26/HER2 tumor-bearing mice. However, IFN-γ had no effect on tumor progression in the CT26/HER2 model, as determined by an IFN-γ knockout assay. 4T1.2/HER2 tumor-bearing mice displayed CTL activity in response to HER263-71 but not to HER2342-350, whereas no such induction was observed in CT26/HER2 tumor-bearing mice. When 4T1.2/HER2 cell-challenged mice were depleted of CD8+ T cells, they lost their tumor-regressing activity, suggesting an antitumor role of HER263-71-specific CD8+ CTLs in the control of this tumor type. CT26/HER2 cells also expressed CD80. However, CD80-transfected 4T1.2/HER2 and CD80-non-expressing CT26/HER2 cells failed to alter their tumorigenicity, suggesting no role of CD80 in tumor control. Despite increased levels of myeloid-derived suppressor cells in the tumor, they were not associated with tumor progression in the CT26/HER2 model, as determined by a cell depletion assay. Overall, these data show that, contrary to CT26/HER2 tumors, 4T1.2/HER2 tumors regress via the induction of HER263-71-specific CD8+ CTLs and that CD80 is not associated with the regression of these tumors.

Diagnostic usefulness of the GenoType MTBDRplus assay for detecting drug-resistant tuberculosis using AFB smear-negative specimens with positive TB-PCR result.

BACKGROUND: The aim of this study was to evaluate the diagnostic accuracy of the GenoType MTBDRplus assay in detecting drug-resistant tuberculosis (DR-TB) by using acid-fast bacilli (AFB) smear-negative specimens with positive TB-PCR results. METHODS: The MTBDRplus assay was performed with 2 different categories of 117 samples, including AFB smear-positive specimens (n = 53) and AFB smear-negative specimens (n =64), which exhibited positive TB-PCR results, at a single institution. The results were retrospectively compared with the results of the phenotypic drug susceptibility test (DST), for reference. RESULTS: A total of 105 tests were finally analyzed. Of these, 54 tests were conducted using AFB smear-negative specimens with positive TB-PCR results. The MTBDRplus assay for these 54 samples demonstrated a sensitivity of 100%, specificity of 98%, positive predictive value (PPV) of 75%, and negative predictive value (NPV) of 100% in detecting rifampicin resistance. With these same species, the sensitivity, specificity, PPV, and NPV values for the MTBDRplus assay were 83.3%, 97.9%, 83.3%, and 97.9%, respectively, for the detection of isoniazid resistance. The overall correlation between the MTBDRplus assay and phenotypic DST demonstrated excellent agreement for detection of rifampicin resistance (κ = 0.847) and for detection of INH resistance (κ = 0.812), respectively. CONCLUSIONS: The MTBDRplus assay can be used effectively even on AFB smear-negative specimens from TB patients, when the TB-PCR is positive. This result might help clinicians to manage patients with suspected DR-TB in difficult situations.

Antioxidative properties of ginsenoside Ro against UV-B-induced oxidative stress in human dermal fibroblasts.

Ginsenoside Ro (Ro), an oleanolic acid-type ginsenoside, exhibited suppressive activities on reactive oxygen species (ROS) and matrix metalloproteinase-2 (MMP-2) elevation in UV-B-irradiated fibroblasts. Ro could overcome the reduction of the total glutathione (GSH) contents in UV-B-irradiated fibroblasts. Ro could not interfere with cell viabilities in UV-B-irradiated fibroblasts. Collectively, Ro possesses a potential skin anti-photoaging property against UV-B radiation in fibroblasts.

DNA vaccines, electroporation and their applications in cancer treatment.

Numerous animal studies and recent clinical studies have shown that electroporation-delivered DNA vaccines can elicit robust Ag-specific CTL responses and reduce disease severity. However, cancer antigens are generally poorly immunogenic, requiring special conditions for immune response induction. To date, many different approaches have been used to elicit Ag-specific CTL and anti-neoplastic responses to DNA vaccines against cancer. In vivo electroporation is one example, whereas others include DNA manipulation, xenogeneic antigen use, immune stimulatory molecule and immune response regulator application, DNA prime-boost immunization strategy use and different DNA delivery methods. These strategies likely increase the immunogenicity of cancer DNA vaccines, thereby contributing to cancer eradication. However, cancer cells are heterogeneous and might become CTL-resistant. Thus, understanding the CTL resistance mechanism(s) employed by cancer cells is critical to develop counter-measures for this immune escape. In this review, the use of electroporation as a DNA delivery method, the strategies used to enhance the immune responses, the cancer antigens that have been tested, and the escape mechanism(s) used by tumor cells are discussed, with a focus on the progress of clinical trials using cancer DNA vaccines.

Stereoselective suppressive effects of protopanaxadiol epimers on UV-B-induced reactive oxygen species and matrix metalloproteinase-2 in human dermal keratinocytes.

This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.

Skin anti-photoaging properties of ginsenoside Rh2 epimers in UV-B-irradiated human keratinocyte cells.

Ginseng, one of the most widely used herbal medicines, has a wide range of therapeutic and pharmacological applications. Ginsenosides are the major bioactive ingredients of ginseng, which are responsible for various pharmacological activities of ginseng. Ginsenoside Rh2, known as an antitumour ginsenoside, exists as two different stereoisomeric forms, 20(S)-ginsenoside Rh2 [20(S)-Rh2] and 20(R)-ginsenoside Rh2 [20(R)-Rh2]. This work aimed to assess and compare skin anti-photoaging activities of 20(S)-Rh2 and 20(R)-Rh2 in UV-B-irradiated HaCat cells. 20(S)-Rh2, but not 20(R)-Rh2, was able to suppress UV-B-induced ROS production in HaCat cells. Both stereoisomeric forms could not modulate cellular survival and NO level in UV-B-irradiated HaCat cells. Both 20(S)-Rh2 and 20(R)-Rh2 exhibited suppressive effects on UV-B-induced MMP-2 activity and expression in HaCat cells. In brief, the two stereoisomers of ginsenoside Rh2, 20(S)-Rh2 and 20(R)-Rh2, possess skin anti-photoaging effects but possibly in different fashions.

Gamibojungikki-tang decreases immobility time on the forced swimming test and increases interferon-gamma production from MOLT-4 cells.

Gamibojungikki-tang (GBIT) has been used for the purpose of development of physical strength in Korea. We investigated the anti-immobility effect of GBIT on the forced swimming test (FST) and then measured the blood biochemical parameters related to fatigue, glucose (Glc); blood urea nitrogen (BUN); lactic dehydrogenase (LDH); creatine kinase (CK) and total protein (TP). GBIT (0.01, 0.1, 1 g/kg) was orally administered to mice for 7 days. After 7 days, the immobility time was significantly decreased in the GBIT-administration group (105.0+/-12.1 s for 1 g/kg) in comparison with the control group (152.3+/-16.2 s). The contents of Glc and TP in the blood serum were significantly increased in GBIT-administration group (1g/kg) compared with control group, while LDH was significantly decreased. Surface phenotyping of spleen cells by FACS analysis revealed an increasing tendency of CD4+ and CD8+ number, without statistical significance. In addition, GBIT (0.01-1 mg/ml) increased the interferon-gamma and interlukin-2 levels in MOLT-4 T-cells. These results suggest that GBIT may be useful in the immune function improvement.

Anti-inflammatory activity of Korean folk medicine 'Sabaeksan'.

Sabaeksan has been used for prevention and treatment of bronchial asthma and allergic asthma in Korea. To investigate the biological effect of Sabaeksan, we examined the effect of Sabaeksan on the phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-induced pro-inflammatory cytokines secretion in human mast cell line HMC-1 cells. Sabaeksan by itself had no effect on viability of HMC-1 cells. The effects of Sabaeksan on the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 from HMC-1 were evaluated with enzyme-linked immunosorbent assay. Sabaeksan (1 mg/ml) inhibited PMA plus A23187-induced TNF-alpha, IL-6, and IL-8 secretion by 43.86+/-5.26%, 56.39+/-3.65%, and 63.48+/-2.54%, respectively. Sabaeksan also inhibited the nuclear factor-kappa B (NF-kappaB) activation and IkappaBalpha degradation. Taken together, these results suggest that Sabaeksan inhibits the secretion of pro-inflammatory cytokines in HMC-1 cells through blockade of NF-kappaB activation.

Inhibitory effect of Gamibojungikgitang extract on mast cell-mediated allergic reaction in murine model.

PURPOSE: Gamibojungikgitang (GBIT) is an Oriental herbal prescription medication, which has been commonly used to treat allergic rhinitis in far Eastern countries including Korea, China, and Japan. Additionally, GBIT effectively treats ovarian cysts and improves ovarian functions by regulating both endocrine and metabolic activities. However, it has not been cleared how it prevents allergic diseases in experimental model. Here we report the effect of GBIT on mast cell-mediated allergic reactions. METHODS: The anti-anaphylactic effect of GBIT extract was studied against compound 48/80-induced systemic anaphylactic shock model in mice. Rat Peritoneal Mast Cells (RPMCs) were used to investigate the effect of GBIT extract on histamine release induced by compound 48/80. Passive cutaneous anaphylaxis activated by anti-dinitrophenyl IgE was used to know the effect of GBIT extract. In addition, human mast cell line HMC-1 cells culture supernatants that GBIT extract pretreated were assayed for IL-6 protein levels by enzyme-linked immunosorbent assay method. RESULTS: GBIT extract dose dependently inhibited compound 48/80-induced systemic anaphylactic shock. When GBIT extract was given as pretreatment at concentrations ranging 0.01-1 mg/ml, the histamine release from rat peritoneal mast cells induced by compound 48/80 was reduced in a dose-dependent manner. GBIT extract also inhibited passive cutaneous anaphylaxis activated by anti-dinitrophenyl IgE. In addition, GBIT extract inhibited phorbol 12-myristate 13-acetate + A23187-induced interleukin-6 secretion from human mast cell line HMC-1 cells. CONCLUSION: These results suggest a potential role for GBIT extract as a source of anti-anaphylactic agents for allergic disorders.

The Oriental medicine "Cool-Cool (Cool-X-A)" inhibits inflammatory cytokine production and migration in mast cells.

Plant medications have been applied to treat pains from various types of arthritis in Korea. Rheumatoid arthritis (RA) is well known to be a chronic autoimmune/inflammatory disease that leads to progressive joint damage and cartilage destruction. Accumulation and activation of mast cells have been demonstrated in rheumatoid synovial tissue. Because infiltrated mast cells and their mediators may contribute to the initiation and progression of the inflammatory process and matrix degradation of RA, we tested the inhibitory effects of "Cool-Cool" (CC, Cool-X-A), an Oriental medication, on the production and migration of major inflammatory cytokines in mast cells. CC was treated in vitro before activation of human mast cell line (HMC-1) with phorbol 12-myristate 13-acetate, and the cytotoxicity of CC was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. CC had no cytotoxic effects on HMC-1 cell viability. The inhibitory effects on cytokine production were monitored by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR). CC inhibited not only the secretion but also the expression of TNF-alpha and IL-8 in HMC-1 cells. CC also suppressed migration of mast cells induced by stem cell factor. These findings may help in understanding the mechanism of action of this herbal medication, leading to the control of mast cells in inflammatory conditions like RA.

Polymorphism of the tumor necrosis factor alpha gene and waist-hip ratio in obese Korean women.

A number of candidate genes have been in implicated in the pathogenesis of obesity in humans. Tumor necrosis factor-alpha (TNFalpha) is expressed primarily in adipocytes, and elevated levels of this cytokine have been linked to obesity and insulin resistance. Recently, the A allele of a polymorphism at position -308 in the promoter region of TNFalpha (G-308A) has been shown to increase transcription of the gene in adipocytes. We therefore designed this study to test whether obese and non-obese subjects differ in terms of TNFalpha genotype distribution. We also investigated whether the genotypes affect anthropometric parameters, such as body mass index (BMI). The study included 153 obese healthy women and 82 non-obese women. Total fat mass and percent body fat were determined by dual-energy X-ray absorptiometry. Genomic DNA was extracted and used for NcoI restriction fragment length polymorphism-based genotyping of TNFalpha. No differences were observed in allele and genotype frequencies between obese and non-obese women, and no association of TNFalpha polymorphism with BMI was observed for genotype in the obese women. In addition, age, percent body fat, BMI, and cholesterol levels did not vary with TNFalpha genotype. However, waist-to-hip ratio (WHR) was significantly lower in subjects with TNFalpha GA or AA genotypes (0.94 +/- 0.07 vs. 0.92 +/- 0.03, P < 0.005). These results indicate that polymorphism at position -308 of the TNFalpha promoter is not a significant factor for BMI, but affects WHR in obese healthy Korean women.