Search

Androgenetic alopecia is a common disease that occurs in both men and women. Several approved medications have been used to treat this condition, but they are associated with certain side effects. Therefore, use of extracts derived from natural products, such as Siberian sturgeon (Acipenser baerii), and the regulation of the gut microbiota have become important topics of research. Sturgeon is known for its high nutritional value and anti-inflammatory properties; however, its effects on androgenetic alopecia and gut microbiota remain uncharacterized. Here, we aimed to investigate whether solubilized sturgeon oil (SSO) promotes hair growth and regulates the gut microbiome. C57BL/6 mice were divided into four groups. Three groups received topical applications of distilled water, SSO, or minoxidil, and one group was orally administered SSO. Each treatment was administered over 4 weeks. Histopathological analysis revealed a significant increase in follicle number (p < 0.001) and follicle diameter (p < 0.05). Immunohistochemical analysis revealed upregulation of β-catenin and ERK-1, markers involved in hair growth-promoting pathways. Furthermore, microbiome analysis revealed that the reduced gut microbiota was negatively correlated with these markers. Our findings indicate that oral administration of SSO promotes hair growth and regulates the abundance of hair growth-promoting gut microbiota. [ABSTRACT FROM AUTHOR]

The RING domain of MUL1 (RINGMUL1) alone mediates ubiquitylation of the p53‐transactivation domain (TADp53). To elucidate the mechanism underlying the simultaneous recruitment of UBE2D2 and the substrate TADp53 by RINGMUL1, we determined the complex structure of RINGMUL1:UBE2D2 and studied the interaction between RINGMUL1 and TADp53 in the presence of UBE2D2–UB thioester (UBE2D2~UB) mimetics. The RINGMUL1‐binding induced the closed conformation of UBE2D2S22R/C85S–UBK48R oxyester (UBE2D2RS–UBROE), and strongly accelerated its hydrolysis, which was suppressed by the additional N77A‐mutation of UBE2D2. Interestingly, UBE2D2S22R/N77A/C85S–UBK48R oxyester (UBE2D2RAS–UBROE) already formed a closed conformation in the absence of RINGMUL1. Although TADp53 exhibited weak binding for RINGMUL1 or UBE2D2 alone, its binding affinity was enhanced and even further for RINGMUL1:UBE2D2 and RINGMUL1:UBE2D2RAS–UBROE, respectively. The recognition of TADp53 by RINGMUL1 as a complex with UBE2D2~UB is related to the multivalency of the binding events and underlies the ability of RINGMUL1 to ubiquitylate the intrinsically disordered protein, TADp53. [ABSTRACT FROM AUTHOR]

Knowledge of the impact of the gut microbiota on human health has increased, and modulation of the bacterial community is now considered a therapeutic target for various diseases. Certain novel bacterial species have probiotic properties associated with improvement in obesity and related metabolic disorders. The relative abundance of Butyricimonas spp. is correlated with metabolic parameters; however, the physiological role of Butyricimonas in metabolic improvement is unclear. In this study, live and heat-killed Butyricimonas virosa were administered to mice with high-fat diet (HFD)-induced obesity. Both live and heat-killed B. virosa ameliorated HFD-impaired body weight, serum glucose level, insulin resistance, and liver steatosis. Moreover, activation of the glucagon-like peptide-1 receptor (GLP-1R) and peroxisome proliferator-activated receptor α (PPARα) was observed in the liver, and the expression levels of insulin receptor substrate (IRS)-1, IRS-2, Toll-like receptor 5 (TLR5), and zonula occludens-1 (ZO-1) were upregulated in the ileum. Finally, we demonstrated that the effect of B. virosa treatment on glucose regulation may be linked to the upregulation of GLP-1R in the liver and is not a result of colonization of the gut by B. virosa or B. virosa -produced butyrate. Our results provide a rationale for the development of Butyricimonas spp.-based therapeutics and prophylactics for hyperglycemia. [ABSTRACT FROM AUTHOR]

Furthermore, the analysis of the reconstructed human skin (RHS) tissue AD model confirmed that CHI3L1-Ab treatment reduced skin inflammation by inhibiting the CHI3L1/NF- B/ITGA5 axis (Figure 3E-I). After confirming that CHI3L1 regulates AD-related skin inflammation, we examined the therapeutic effect of CHI3L1-antibody (Ab). Furthermore, the inhibition of CHI3L1 by CHI3L1-Ab suppresses AD-like symptoms and inflammatory responses by regulating CHI3L1, the NF- B/ITGA5 axis. [Extracted from the article]

Influenza viruses cause significant morbidity and mortality worldwide. Long-term or frequent use of approved anti-influenza agents has resulted in drug-resistant strains, thereby necessitating the discovery of new drugs. In this study, we found aprotinin, a serine protease inhibitor, as an anti-influenza candidate through screening of compound libraries. Aprotinin has been previously reported to show inhibitory effects on a few influenza A virus (IAV) subtypes (e.g., seasonal H1N1 and H3N2). However, because there were no reports of its inhibitory effects on the other types of influenza viruses, we investigated the inhibitory effects of aprotinin in vitro on a wide range of influenza viruses, including avian and oseltamivir-resistant influenza virus strains. Our cell-based assay showed that aprotinin had inhibitory effects on seasonal human IAVs (H1N1 and H3N2 subtypes), avian IAVs (H5N2, H6N5, and H9N2 subtypes), an oseltamivir-resistant IAV, and a currently circulating influenza B virus. We have also confirmed its activity in mice infected with a lethal dose of influenza virus, showing a significant increase in survival rate. Our findings suggest that aprotinin has the capacity to inhibit a wide range of influenza virus subtypes and should be considered for development as a therapeutic agent against influenza. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a chronic inflammatory skin disease. A hallmark of AD is dry itchy skin that results from defects in the epidermal barrier function. Aloe vera is used widely to promote general health and is administered topically to treat skin conditions such as eczema, burns and wounds. However, effects of A vera on AD were not fully elucidated. In this study, we investigated the oral administration of processed A vera gel (PAG) containing low molecular weight Aloe polysaccharides to treat ovalbumin (OVA)‐induced AD in mice. Oral administration of PAG suppressed total and OVA‐specific IgE production in sera and decreased the epidermal thickness of skin. Numbers of Ki‐67‐positive cells were reduced by PAG treatment. Expression levels of tight junction genes, including those that encode ZO‐1, Claudin‐1 and Claudin‐8, were decreased in AD skin lesions, whereas oral administration of PAG partially restored the expression levels of tight junction genes. In addition, IL‐4 and IL‐17A mRNA transcript levels were reduced in skin lesions after PAG treatment. Taken together, our findings suggest that oral administration of PAG ameliorated AD, normalized tight junction gene expression and suppressed inflammatory cytokines in AD skin. [ABSTRACT FROM AUTHOR]

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C‐terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1‐RING) where the Ub~E2 thioester binds. Unlike most RING‐type E3 enzymes, MUL1‐RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1‐RING has not yet been elucidated. In order to understand this novel ubiquitylation mechanism, it is necessary to determine the three‐dimensional structures of MUL1‐RING and of its complex with the cognate E2 enzyme. Here, Ube2D2 was validated as a functional E2 enzyme for the ubiquitylation of the p53 transactivation domain (p53‐TAD) by MUL1‐RING, and purification and crystallization processes for MUL1‐RING and the MUL1‐RING–Ube2D2 complex are reported. [ABSTRACT FROM AUTHOR]

Dysbiosis of the gut microbiota is a contributing factor for obesity-related metabolic diseases such as hyperglycemia and hyperlipidemia. Pharmacotherapy for metabolic diseases involves the modulation of gut microbiota, which is suggested to be a potential therapeutic target. In this study, the modulation of gut microbiota by statins (cholesterol-lowering drugs: atorvastatin and rosuvastatin) was investigated in an aged mouse model of high-fat diet-induced obesity, and the association between gut microbiota and immune responses was described. Atorvastatin and rosuvastatin significantly increased the abundance of the genera Bacteroides , Butyricimonas , and Mucispirillum. Moreover, the abundance of these genera was correlated with the inflammatory response, including levels of IL-1β and TGFβ1 in the ileum. In addition, oral fecal microbiota transplantation with fecal material collected from rosuvastatin-treated mouse groups improved hyperglycemia. From these results, the effect of statins on metabolic improvements could be explained by altered gut microbiota. Our findings suggest that the modulation of gut microbiota by statins has an important role in the therapeutic actions of these drugs. [ABSTRACT FROM AUTHOR]

We developed spontaneous diet-induced metabolic disease in mice by feeding them a high-fat diet for 23 weeks and administered Aloe QDM complex for 16 weeks to examine its restorative effect on immune disorders and metabolic syndrome. A series of immune functional assays indicated Aloe QDM complex enhanced lymphocyte proliferation and antigen-specific immunity as determined by the restored functions of cytotoxic T lymphocytes (CTL) and IgG production. The elevated serum TNF-α level was also regulated by Aloe QDM complex treatment, which suggested its complex therapeutic potential. As for metabolic phenotypes, oral administration of Aloe QDM complex significantly improved diabetic symptoms, including high fasting glucose levels and glucose tolerance, and distinctly alleviated lipid accumulation in adipose and hepatic tissue. The simultaneous restoration of Aloe QDM complex on metabolic syndrome and host immune dysfunction, especially on the specific CTL killing was first elucidated in our study. [ABSTRACT FROM PUBLISHER]

The effects of spiramycin on adipogenesis and high fat diet (HFD)-induced obesity were investigated. Potential mechanisms contributing to these effects were elucidated. The inhibitory effect of spiramycin on adipocyte differentiation was assessed using 3T3-L1 preadipocyte cells, in which several parameters involved in AMPK signal pathways and lipid metabolism were examined. To further investigate the pharmacological effects of spiramycin in vivo, we examined several obesity-related parameters in HFD-induced obese mice. Spiramycin significantly inhibited preadipocyte differentiation by attenuating intracellular lipid accumulation. Spiramycin also reduced the expression of adipogenic master regulators (PPARγ, C/EBPα, and SREBP1c) and their downstream target genes (FAS, aP2, and GLUT4) in 3T3-L1 cells. In addition, AMPK phosphorylation was increased by spiramycin treatment in 3T3-L1 cells during early differentiation. Notably, HFD-induced obese mice administered spiramycin showed substantial decreases in body weight gain, serum leptin levels, adipose tissue mass, and hepatic lipid accumulation. Moreover, the decreased levels of GPT and GOT in the serum indicated that spiramycin attenuated hepatic injury caused by HFD. Taken together, these results demonstrate for the first time that spiramycin effectively attenuates HFD-induced obesity and hepatic steatosis by inhibiting adipogenesis. [ABSTRACT FROM AUTHOR]

Background Little information is available regarding the precise swine leukocyte antigen ( SLA)-derived immunogenic peptides that are presented in the context of human HLA molecules. Here, we identified SLA-derived immunogenic peptides that are presented in association with human HLA-A2 molecule. Methods The SLA-derived peptides that bind to HLA-A*0201, a representative of the A2 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and the stabilities of complexes formed between peptides and HLA-A*0201 were compared using major histocompatibility complex ( MHC) stabilization assays. The cytotoxic T lymphocyte ( CTL)-inducing activity of the selected peptides was examined in HLA-A*0201-transgenic mice. Results Among 15 candidate peptides synthesized, two peptides, peptide-35 ( YLGPDGLLL) and peptide-43 ( TLICHVDSI), were selected to have high affinity and stability with HLA-A*0201. Examination of the CTL-inducing activity of the two peptides in HLA-A*0201-transgenic mice showed that immunization with peptide-35, but not peptide-43, elicited potent CD8-specific CTL responses. The Peptide-35 is present in non-polymorphic α2 domains of 34 SLA-1 alleles, 18 SLA-2 alleles, and 1 SLA-3 allele. Conclusion This study identifies an immunogenic HLA-A*0201-restricted epitope derived from the SLA, which may be valuable for the development of epitope-specific immunoregulation strategies. [ABSTRACT FROM AUTHOR]

Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patients and tumor-bearing mice, subsequently suppressing the host immune system. MDSCs represent a group of immature myeloid cells expressing CD11b and Gr-1. Here, we show that a Toll-like receptor (TLR) agonist, resiquimod, which binds to TLR7 and TLR8, induces the differentiation of MDSCs into mature myeloid cells. MDSCs were isolated from mice bearing mammary carcinoma 4T1 cells, and the purified MDSCs were cultured in the presence of resiquimod for 5 days. Phenotypic analysis showed that the resiquimod-treated MDSCs differentiated into F4/80 macrophages and CD11c/I-A dendritic cells. Functional analysis showed that the MDSCs also lost their suppressive activity on T cells. Resiquimod-treated MDSCs significantly enhanced the proliferation of T cells that were treated with anti-CD3 and anti-CD28 monoclonal antibodies. These results show that resiquimod induces the differentiation of MDSCs into macrophages and dendritic cells, and also suggest that resiquimod may improve cancer immunotherapy by reducing immunosuppressive MDSCs. [ABSTRACT FROM AUTHOR]

Adiponectin is an adipocyte hormone involved in glucose and lipid metabolism. The aim of this study was to develop a human adiponectin expression system in transgenic silkworm using a human adiponectin expression vector. The silk gland of the silkworm is a highly specialized organ that has the wonderful ability to synthesize and secrete silk protein. To express human adiponectin in the silk gland of transgenic silkworm, targeting vectors pB-A3-adiponectin-IRES-RFP and pB-Ser1-adiponectin-IRES-RFP were constructed and then introduced into the silkworm pupa. The transgenic silkworms were verified by PCR and then generated. The level of adiponectin in the transgenic silkworm was 6-10 ng/50 mg of freeze-dried powder, and western blotting using an antibody against human adiponectin demonstrated a specific band with a molecular weight of 30 kDa in the silkworm. These results showed that human adiponectin introduced into the silkworm genome was expressed successfully on a large-scale. [ABSTRACT FROM AUTHOR]

6-Hydroxy-2,7-dimethoxy-1,4-phenanthraquinone (PAQ) isolated from the tuberous roots of Yam ( Dioscorea batatas) inhibited cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) dependent prostaglandin D (PGD) generation in mouse bone marrow-derived mast cells in a concentration-dependent manner with IC values of 0.08 μM and 0.27 μM, respectively. In the Western blotting with specific anti-COX-2 antibodies, the decrease of the quantity of PGD was accompanied by a decrease in the COX-2 protein level. But PAQ did not affect COX-1 protein level. In addition, this compound inhibited 5-lipoxygenase (5-LOX) dependent production of leukotriene C in a dose-dependent manner, with an IC of 0.032 μM. These results demonstrate that PAQ has a dual COX-2/5-LOX inhibitory activity. This compound also inhibited the degranulation reaction in a dose-dependent manner with an IC of 2.7 μM. Thus, these results suggest that PAQ may be useful in regulating mast cell-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

The effects of intraphagosomal toll-like receptor (TLR) activation on the MHC-restricted presentation of exogenous antigen were examined in dendritic cells (DCs). For phagosomal targeting, nanoparticles containing both a TLR agonist and a model antigen, ovalbumin (OVA), were prepared using biodegradable polymer poly(D,L-lactic acid-co-glycolic acid) and were then opsonized with mouse IgG. After incubating mouse DCs with the nanoparticles, the efficacy of OVA peptide presentation was evaluated using OVA-specific CD8 and CD4 T cells. Inclusion of either the TLR3 agonist poly(I:C) or the TLR9 agonist CpG oligodeoxynucleotides (ODN) significantly increased and prolonged both MHC class I- and class II-restricted OVA presentation. Accordingly, the DCs that phagocytosed the nanoparticles containing poly(I:C) or CpG ODN together with OVA efficiently induced the proliferation of OVA-specific CD8 and CD4 T cells. The potency levels of poly(I:C) and CpG ODN in increasing the MHC-restricted presentation of the exogenous antigen appeared to be similar. A combination of the 2 TLR agonists was synergistic in increasing the MHC class I-restricted, but not the class II-restricted, presentation of exogenous antigen. These results show that IgG-opsonized biodegradable nanoparticles containing both intraphagosomal TLR agonists and antigens can be efficient carrier materials in inducing antigen-specific T cell responses. [ABSTRACT FROM AUTHOR]

We investigated the anticancer activity of 11-hydroxyaclacinomycin X (ID-6105), a novel anthracycline, on weakly doxorubicin (Dox)-resistant SK-OV-3 ovarian cancer cells, and elucidated the relationship between its anticancer activity and accumulation in cells compared with those of Dox. Accumulation of ID-6105 in the cells was time-and concentration-dependent, a result of drug-induced cytotoxicity in the cells. SK-OV-3 cells were preloaded with ID-6105 or Dox for 12 h at concentrations ranging from 100 to 2000 nM and then incubated with drug-free medium for 0–48 h. Cell viability was measured using a proliferation-based assay (XTT assay). The inhibitory effects of ID-6105 on cell viability were more pronounced than those of Dox. The IC50 values of ID-6105 after 24-and 48-h incubation with drug-free medium were 1.58 and 0.084 μM, while those of Dox were 2 and 0.334 μM, respectively. To investigate the relationship between the intracellular levels and the cytotoxic effects of the drugs, we preloaded SKOV-3 cells with ID-6105 or Dox (100–2000 nM) for 12 h and then measured the intracellular levels of drugs by HPLC in drug-free medium for 0–48 h. After preloading the drugs, the intracellular concentrations of ID-6105 at time 0 were 1.3-, 1.8-, and 1.4-fold larger than those of Dox at initial concentrations of 500, 1000, and 2000 nM, respectively. The extent of ID-6105 accumulation in the cells was more pronounced than that of Dox. These findings suggest that ID-6105 effluxed less from the cells than Dox, resulting in its extensive cytotoxicity compared with that of Dox. These results show that accumulation of ID-6105 within tumor cells may be important for the inhibitory effects of this drug in cancer cells. ID-6105 has an antiproliferative effect on SK-OV-3 cells that is due to its cytotoxicity. This effect is more pronounced than that of Dox, and may be attributed to extensive accumulation of ID-6105 in the cells. [ABSTRACT FROM AUTHOR]

A 1:1 mixture of acriflavine (ACF; CAS 8063-24-9) and guanosine is under evaluation in preclinical studies as a possible antitumor agent. Guanosine is known to potentiate the anti-cancer activity of ACF. We therefore investigated the pharmacokinetics of guanosine following administration of the ACF/guanosine mixture in rats. Rats were given guanosine (1 or 5 mg/kg) or ACF/guanosine (2 or 10 mg/kg) by i.v. bolus; or guanosine (3 or 15 mg/kg) or ACF/guanosine (6 or 30 mg/kg) by i.m. injection. We found that guanosine was rapidly cleared from the blood and transferred to tissues after i.m. administration of ACF/guanosine. The mean plasma half-lives (t1/2) at the α and β phases were 0.091 and 6.86 h, or 0.09 and 7.51 h at a dose of 1 or 5 mg/kg guanosine, respectively. ACF had no effect on the plasma disappearance of guanosine following either i.v. bolus or i.m. administration of the combination mixture. Moreover, the ACF combination with guanosine did not significantly alter the values of MRT, Vdss, and CLt of guanosine. Guanosine exhibited linear pharmacokinetics over the dose range from 1 to 5 mg/kg for i.v. doses and 3 to 15 mg/kg for i.m. doses. The bioavailability of guanosine after i.m. administration was 84% for 3 mg/kg dose and 88% for 15 mg/kg dose. ACF had no effects on biliary and urinary excretion of guanosine after i.m. administration. The cumulative amount of guanosine in urine after i.m. administration was about 5-fold larger than that in bile, indicating that guanosine is mostly excreted into the urine. Guanosine was widely distributed in all tissues examined in this study, but was most highly concentrated in the kidney after i.m. administration, followed by slow excretion to bile or urine. ACF had no effect on the tissue distribution of guanosine following i.m. administration. These characterizations of the pharmacokinetics of guanosine after administration of the ACF/guanosine combination will be useful in providing preclinical and clinical bases for the potential application of this combination to the treatment of cancer. [ABSTRACT FROM AUTHOR]

The effects of asimilobine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and l-DOPA-induced cytotoxicity in PC12 cells were investigated. Asimilobine at concentration ranges of 0.05-0.2 μM showed a significant inhibition of intracellular dopamine levels for 24 h in a concentration-dependent manner with an IC50 value of 0.13 μM. Asimilobine at 0.15 μM inhibited tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC) activities at 24 h (73.2% inhibition of TH activity): the inhibition of TH activity was stronger and longer than that of AADC activity. Asimilobine also decreased TH mRNA levels and intracellular cyclic AMP levels, but not the basal Ca2 + concentrations. In addition, asimilobine at 0.05-5.0 μM, but not 10 μM, did not alter cell viability toward PC12 cells. A non-cytotoxic asimilobine (0.15 μM) associated with l-DOPA (20, 50, and 100 μM) for 24 h inhibited l-DOPA-induced increases in dopamine levels and enhanced l-DOPA-induced cell death when compared with l-DOPA alone. These results suggest that asimilobine inhibits dopamine biosynthesis by mainly reducing the TH activity and TH mRNA expression, and enhances l-DOPA-induced cytotoxicity in PC12 cells. [ABSTRACT FROM AUTHOR]

Auranofin (AF), an orally administered, gold-based, anti-arthritic agent, has emerged as a clinically useful therapeutic drug for the treatment of rheumatoid arthritis. In the present study, we examined the effects of AF on major histocompatibility complex (MHC)-restricted antigen presentation in dendritic cells (DCs), which are the most important accessory cells for the induction of T cell responses. A mouse dendritic cell line, DC2.4 cells, and DCs that were generated from mouse bone marrow cells by culturing with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 were each pretreated with AF for 2 hr, and then incubated with ovalbumin (OVA). After the 2-hr incubation, the DCs were fixed, and the amounts of OVA peptide-H-2Kb complexes were assessed using OVa-specific CD8+ T cells. AF inhibited MHC class I-restricted presentation of exogenous OVA. This inhibitory activity of AF appeared to be due not only to the inhibition of the phagocytic activity of DCs, but also to the suppression of MHC molecule expression on DCs. AF also inhibited MHC class II-restricted presentation of exogenous OVA. These results show that AF exerts immunosuppressive activity at least in part by inhibiting MHC-restricted antigen presentation in professional antigen-presenting cells. [ABSTRACT FROM AUTHOR]

We have reported that ginseng total saponin (GTS) inhibited the development of physical and psychological dependence on morphine. However, the possible molecular mechanisms of GTS are unclear. Therefore, this study was undertaken to understand the possible molecular mechanism of GTS on the inhibitory effects of morphine-induced dependence. It has been reported that the up-regulated cAMP pathway in the LC of the mouse brain after repeated administration of morphine contributes to the feature of withdrawals. GTS inhibited up-regulation of cAMP pathway in the LC after repeated administration of morphine in this experiment. GTS inhibited cAMP levels and protein expression of protein kinase A (PKA). In addition, GTS inhibited the increase of cAMP response element binding protein (CREB) phosphorylation. Therefore, we conclude that the inhibitory effects of GTS on morphine-induced dependence might be mediated by the inhibition of cAMP pathway. [ABSTRACT FROM AUTHOR]

Auranofin (AF), a gold compound, is an orally active therapeutic agent used to treat rheumatoid arthritis (RA), a self-perpetuating inflammatory disease. RA is characterized by autoimmune-mediated proliferation of synovial cells that leads to inflammation, pain, and swelling in most major joints. However, the mechanism as to how AF relieves RA symptoms has not been fully elucidated. The object of this study was to examine the ability of AF to immunomodulate macrophages as antigen presenting cells (APCs). Macrophages are recognized as playing an important role in the pathogenesis of RA, in that there is a relative abundance of macrophage-derived cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in rheumatoid synovium. In this work, we tested whether AF (2.5–20 mM) could inhibit inflammatory activity in the macrophage cell line RAW 264.7. AF decreased production of nitric oxide (NO) and the pro-inflammatory cytokines, TNF-α, IL-1β and IL-6 in macrophages. Furthermore, AF inhibited cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production in a concentration-dependent manner. In conclusion, these findings may provide an explanation for the clinical effects of AF in patients with RA. [ABSTRACT FROM AUTHOR]

Mizoribine (MZR) has been shown to possess immunosuppressive activity that selectively inhibits the proliferation of lymphocytes by interfering with inosine monophosphate dehydrogenase. The efficacy of MZR is not only in patients who have had renal transplantation, but also in patients with rheumatoid arthritis (RA), lupus nephritis, and primary nephritic syndrome. Because the exact mechanism of its immunosuppressive action is not clear, the object of this study was to examine the ability of MZR to regulate the antigen presenting cells (APCs), dendritic cells (DCs). In this work, we tested whether MZR (1∼10 μg/mL) could inhibit the crosspresentation of DCs. DC2.4 cells (H-2Kb) or bone marrow-derived DCs (BM-DCs) generated from BM cells of C57BL/6 mouse (H-2Kb) were cultured in the presence of MZR with OVA-microspheres, and the amount of OVA peptide-class I MHC complexes was measured by a T cell hybridoma, B3Z, that recognizes OVA (257–264: SIINFEKL)-H-2Kb complex and expresses-galactosidase. MZR profoundly inhibited the expression of SIINFEKL-H-2Kb complexes. This inhibitory activity of MZR appeared to affect the phagocytic activity of DCs. MZR also decreased IL-2 production when we examined the effects of MZR on CD4+ T cells. These results provide an understanding of the mechanism of immunosuppressive activity of MZR on the inhibition of MHC-restricted antigen presentation and phagocytic activity in relation to their actions on APCs. [ABSTRACT FROM AUTHOR]

Aerial parts of Artemisia asiatica (Compositae) have been traditionally used as an oriental medicine for the treatment of inflammatory and ulcerogenic diseases. In the present study, artemisolide was isolated as a nuclear factor (NF)-κB inhibitor from A. asiatica by activity-guided fractionation. Artemisolide inhibited NF-κB transcriptional activity in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7 with an IC50 value of 5.8 μM. The compound was also effective in blocking NF-κB transcriptional activities elicited by the expression vector encoding the NF-κB p65 or p50 subunits bypassing the inhibitory kB degradation signaling NF-κB activation. The macrophages markedly increased their PGE2 and NO production upon exposure to LPS alone. Artemisolide inhibited LPS-induced PGE2 and NO production with IC50 values of 8.7 μM and 6.4 μM, respectively, but also suppressed LPS-induced synthesis of cyclooxygenase (COX)-2 or inducible NO synthase (iNOS). Taken together, artemisolide is a NF-κB inhibitor that attenuates LPS-induced production of PGE2 or NO via down-regulation of COX-2 or iNOS expression in macrophages RAW 264.7. Therefore, artemisolide could represent and provide the anti-inflammatory principle associated with the traditional medicine, A. asiatica. [ABSTRACT FROM AUTHOR]

Abstract: Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest. [Copyright &y& Elsevier]

Betulinic acid (BA), a pentacyclic triterpene isolated from Lycopus lucidus, has been reported to be a selective inducer of apoptosis in various human cancer and shown anti-inflammatory and immunomodulatory properties. We postulated that BA modulates the immunomodulatory properties at least two groups of protein mediators of inflammation, interlukin-1β (IL-1β) and the tumor necrosis factor-a (TNF-α) on the basis of the critical role of the monocytes and tissue macrophages in inflammatory and immune responses. TNF-α and IL-1β were produced by BA in a dose dependent manner at concentration of 0.625 and 10 μg/mL. The production of NO associated with iNOS was inhibited when treated with LPS at the concentration of 2.5 to 20 μg/mL of BA whereas COX-2 expression was decreased at 2.5 to 20 μg/mL. These modulations of inflammatory mediators were examined in LPS-stimulated RAW 264.7 cells and peritoneal macrophages. The morphology of macrophage was also examined and enhanced surface CD 40 molecule was expressed when treated BA at 0.625–5 ¼g/mL with or without LPS. Furthermore, BA (20 μg/mL) enhanced apoptosis by producing DNA ladder in the RAW 264.7 cells. Our results indicated that BA induced activation of macrophage and pro-inflammatory cytokines. This may provide a molecular basis for the ability of BA to mediate macrophage, suppress inflammation, and modulate the immune response. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) is a neurotropic DNA virus which has latency in human. In this study, we isolated various HSV-1 strains, named KHS, from the skin lesions of Korean patients and characterized the specific features of each strain. We found that KHS strains produced small, cell associated and nonsyncycial plaques in Vero cells. We classified KHS strains into two substrains, KHS 1 which had highly condensed plaques and KHS 2 which had less condensed plaques. Since gD protein of HSV-1 plays important roles in viral plaque formation, we determined the nucleotide sequences of gD genes of KHS strains. According to deduced amino acid sequences of gD protein in KHS strains compared with prototype strains KOS and F, we found that gD of KHS strains have more putative O-glycosidic sites, serine in KHS 1 and threonine in KHS 2, respectively. To find out the establishment of viral latency, we infected each virus strain into eyes of mice and carried out trigerminal ganglia explanting experiment. We found that both KHS strains established latent infections stably just as did the prototype KOS and F strains. The eye swab experiments were carried out to check the viral replication in vivo. KHS 1 exhibited a longer shedding time in eyes of mice. We also found that KHS 1 has a higher neurotropic affinity by determining the time it took for the virus to reach the trigerminal ganglia from the eyes. Currently, we are studying the possible mechanism of high neuroinvasiveness of KHS 1 strain. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is remarkably efficient at establishing chronic infection. One of the reasons for this appears to be the suppression of the accessory cell function of professional antigen presenting cells. In the present study, the immunosuppressive activity of HCV protein was examined on dendritic cells (DCs) generated from mouse bone marrow progenitor cells in vitro. We found that the DCs forced to express HCV protein have defective allostimulatory ability. DCs expressing HCV protein were phenotypically indistinguishable from normal DCs. However, they were unable to produce IL-12 effectively when stimulated with lipopolysaccharide. The functional domain of the HCV protein essential for immunosuppression was determined using a series of NH2-and C-terminal deletion mutants of HCV core protein. We found that amino acid residues residing between the 21st and the 40th residues from the NH2-terminus of HCV core protein are required for immunosuppression. These findings suggest that HCV core protein suppresses the elicitation of protective Th1 responses by the inhibition of IL-12 production by DCs. [ABSTRACT FROM AUTHOR]

The herb, Chrysanthemum zawadskii var, latilobum commomly known as Gu-Jul-Cho in Korea, used in traditional medicine to treat pneumonia, bronchitis, cough, common cold, pharyngitis, bladder-related disorders, gastroenteric disorders, and hypertension. Linarin is the main active compound and the biological mechanisms of its activity are unclear. It is believed that effects of this herb may be exerted through the pluripotent effectors of linarin due to its ability to treat a variety of afflictions. In this study, the effects of linarin on the mouse macrophages cell line, RAW 264.7, were investigated. It was found that linarin could activate macrophages by producing cytokines. Monocytes and tissue macrophages produce at least two groups of protein mediators of inflammation, interleukin 1 (IL-1) and the tumor necrosis factor (TNF). Recent studies have shown that TNF and IL-1 modulate the inflammatory function of endothelial cells, leukocytes, and fibroblasts. TNF-α production by macrophages treated with linarin occured in a dose dependent manner. However, IL-1 production was largely unaffected by this natural product. This study demonstrated the ability of linarin to activate macrophages both directly and indirectly. Linarin also affect both cytokine production and nitric oxide inhibition, in addition to the expression of some surface molecules. Nitric oxide (NO), derived from L-argin-ine, is produced by two forms(constitutive and inducible) of nitric oxide synthase (NOS). The NO produced in large amounts by inducible NOS is known to be responsible for the vasodilation and hypotension observed in septic shock. Linarin was found to inhibit NO production in the LPS-activated RAW 264.7 cells. Linarin may be a useful candidate as a new drug for treating endotoxemia and the inflammation accompanied by NO overproduction. The linarin-treated total lymphocytes exhibited cytotoxicity in a dose dependent manner between 20 ng/ml and 40 (üg/ml. These results suggest that linarin may function through macrophage activation. [ABSTRACT FROM AUTHOR]

A thymic stromal cell line, TFGD, was established from a thymic tumor mass developed spontaneously in p53 knock out. mouse, and was found to produce cytokines that could induce bone marrow hematopoietic stem cells (HSCs) to differentiate into macrophages. The cytokines produced by the TFGD line were assessed by immunoassays. High level of macrophagecolony stimulating factor (M-CSF) and interleukin (IL)-6 was detected in the TFGD-culture supernatant, whereas granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-3, IL-4, IL-5, IL-13, or interferon (IFN)-γ was undetectable. Blocking experiments showed that anti-M-CSF monoclonal antibody could neutralize the differentiation-inducing activity shown by the TFGD-culture supernatant. Dot blot analysis of the total RNA isolated from the cultured fetal thymic stromal cells showed that M-CSF transcripts were expressed in the normal thymus. These observations, together with the earlier finding, that M-CSF plus IL-6 is the optimal combination of cytokines for the induction of macrophage differentiation from HSCs in vitro, may indicate that thymic macrophages could be generated within the thymus by cytokines involving M-CSF. [ABSTRACT FROM AUTHOR]

Background: Local delivery agents (LDA) have the advantage of delivering the antibiotics at high concentrations to the targeted sites. However, the constant flow of gingival crevicular fluids and saliva may restrict their efficacy. Therefore, the drug sustainability and pharmacodynamic properties of any proposed LDA should be evaluated. Methods: Four dental implants were placed unilaterally in the edentulous mandible of six beagle dogs. Peri-implantitis were experimentally induced using silk-ligatures. Each implant was randomly allocated to receive one of the following four treatments: (i) MC (Chitosan-alginate (CA) minocycline), (ii) MP (CA-without minocycline), (iii) PG (Polyacrylate-glycerin minocycline), and (iv) Control (mechanical debridement only). Mechanical therapies and LDAs were administered into the gingival sulcus two times at a 4-week interval. Drug sustainability as well as clinical, radiographical, and immunohistochemical (IHC) analyses were conducted to evaluate the efficacies of treatments. Results: Reduced mean probing depth was observed in all of the test groups after the second delivery. A minimal marginal bone level change was observed during the treatment period (MP (−0.06 ± 0.53 mm) to PG (−0.25 ± 0.42 mm)). The distribution of IHC cell marker analysis of all targeted antibodies ranged from 6.34% to 11.33%. All treatment outcomes between the test groups were comparable. A prolonged retention of LDA was observed from CA microspheres (MC and MP) at both administrations (p < 0.017) and prolonged sustainability of bacteriostatic effect was observed from MC compared to PG after the second administration (p < 0.05). Conclusions: Prolonged retention of CA microspheres was observed and the longer bacteriostatic effect was observed from the MC group. Mechanical debridement with adjunct LDA therapy may impede peri-implantitis progression, however, prolonged drug action did not lead to improved treatment outcome. [ABSTRACT FROM AUTHOR]

Background: The objective of this is preclinical investigation was to evaluate the differential drug sustainability and pharmacodynamic properties of two local minocycline microsphere carriers: chitosan-coated alginate (CA) and poly(meth)acrylate-glycerin (PG). Methods: Four dental implants were placed unilaterally in the edentulous mandible of six beagle dogs. Each implant was randomly assigned to receive one of the following four treatments: (i) CA (CA-based minocycline), (ii) placebo (CA substrate without minocycline), (iii) PG (PG-based minocycline) and (iv) control (mechanical debridement only). After inducing peri-implant mucositis, the randomly assigned treatments were administered into the gingival sulcus twice at a 4-week interval using a plastic-tipped syringe. Drug sustainability and pharmacodynamic (clinical, radiographical and cell marker intensity) evaluations were performed after each administration. Results: The CA microspheres remained longer around the healing abutment compared to the PG microspheres at both administrations and a longer bacteriostatic effect was observed from CA (7.0 ± 5.7 days) compared to PG (1.2 ± 2.6 days). The efficacy of the applied therapies based on clinical, radiographical and histological analyses were comparable across all treatment groups. Conclusions: CA microspheres showed longer carrier and bacteriostatic effect sustainability when compared to PG microspheres, however, longer drug sustainability did not lead to improved treatment outcomes. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family. ZIKV infection has been associated with neurological complications such as microcephaly in newborns and Guillain-Barré syndrome in adults; thus, therapeutic agents are urgently needed. Statins are clinically approved for lowering cholesterol levels to prevent cardiovascular disease but have shown potential as antiviral drugs. In this study, we explored the possibility of utilizing statins as anti-ZIKV drugs. We found that, generally, lipophilic statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, and simvastatin) could reduce ZIKV production in vitro and result in smaller foci of infection. Time-of-drug-addition assay revealed that early treatment with statins is more beneficial than late treatment; however, statins could not completely inhibit the entry stage of ZIKV infection. Furthermore, individual lipophilic statins differed in anti-ZIKV capacity, with fluvastatin being the most efficient at low concentrations. Taken together, this study shows that statins or their derivatives have the potential to be used as anti-ZIKV therapeutic agents. [ABSTRACT FROM AUTHOR]

Park C-S, Kim K-H, Im S-A, Song S, Lee C-K. Identification of HLA-DR4-restricted immunogenic peptide derived from xenogenic porcine major histocompatibility complex class I molecule. Xenotransplantation 2012; 19: 317-322. © 2012 John Wiley & Sons A/S. Abstract: Indirect recognition of xenoantigens has been implicated as the major mechanism underlying xenospecific CD4+ T-cell activation in chronic rejection. We identified swine leukocyte antigen (SLA)-derived immunogenic peptides that are presented in the context of human HLA-DR4 molecules. The SLA class I-derived peptides that bind HLA-DRB1*0401, a representative of the DR4 supertype, were predicted using a computer-assisted algorithm. The candidate peptides were synthesized, and their binding capacities to HLA-DRB1*0401 were compared in a competitive ELISA using biotinylated hemagglutinin reporter peptides [HA307-319]. Peptide-11 (LRSWTAADTAAQISK) was determined to exhibit the most potent binding capacity to HLA-DRB1*0401 in vitro and thus selected for in vivo immunization. Immunization of HLA-DRB1*0401-transgenic mice with peptide-11 elicited potent CD4+ Th1 responses. Peptide-11 shares homology to α2 domains of three SLA-1 alleles, six SLA-2 alleles, and 14 SLA-3 alleles. Thus, this study has important implications not only for the identification of an immunogenic indirect epitope shared by diverse SLA class I alleles, but also for the development of epitope-specific immunoregulation strategies. [ABSTRACT FROM AUTHOR]