Dissolvable microneedles based on Panax notoginseng polysaccharide for transdermal drug delivery and skin dendritic cell activation.

This work explored the feasibility of using biological polysaccharide to fabricate dissolvable microneedles (MNs) for the purpose of transdermal drug delivery and skin dendritic cell (DC) activation. Panax notoginseng polysaccharide (PNPS), a naturally derived immunoactive macromolecule, was used to fabricate dissolvable MNs. The prepared PNPS MNs showed a satisfactory mechanical strength and a skin penetration depth. By Franz diffusion cell assay, the PNPS MNs demonstrated a high transdermal delivery amount of model drugs. Furthermore, with the assistance of MNs, PNPS easily penetrated across the stratum corneum and target ear skin DCs, activating the maturation and migration of immunocytes by increasing the expressions of CD40, CD80, CD86, and MHC II of skin DCs. Consequently, the matured DCs migrated to the auricular draining lymph nodes and increased the proportions of CD4+ T and CD8+ T cells. Thus, PNPS might be a promising biomaterial for transdermal drug delivery, with adjuvant potential.

Yin Yang-1 suppresses CD40 ligand-CD40 signaling-mediated anti-inflammatory cytokine interleukin-10 expression in pulmonary adventitial fibroblasts by promoting histone H3 tri-methylation at lysine 27 modification on interleukin-10 promoter.

During the pathogenesis of early pulmonary arterial hypertension (PAH), pulmonary arterial adventitial fibroblast act as an initiator and mediator of inflammatory processes that predispose vessel walls to excessive vasoconstriction and pathogenic vascular remodeling. Emerging studies report that Yin Yang-1 (YY-1) plays important roles in inflammatory response and vascular injury. Our recent study finds that activation of CD40 ligand (CD40L)-CD40 signaling promotes pro-inflammatory phenotype of pulmonary adventitial fibroblasts. However, whether YY-1 is involved in CD40L-CD40 signaling-triggered inflammatory response in pulmonary adventitial fibroblasts and its underlying mechanism is still unclear. Here, we show that soluble CD40L (sCD40L) stimulation promotes YY-1 protein expression and suppresses anti-inflammatory cytokine, interleukin 10 (IL-10) expression in pulmonary adventitial fibroblasts, while YY-1 knockdown prevents sCD40L-mediated reduction of IL-10 expression via enhancing IL-10 gene transactivation. Further, we find that sCD40L stimulation significantly increases histone H3 tri-methylation at lysine 27 (H3K27me3) modification on IL-10 promoter in pulmonary adventitial fibroblasts, and YY-1 knockdown prevents the effect of sCD40L on IL-10 promoter by reducing the interaction with enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, binding to IL-10 promoter. Moreover, we find that sCD40L stimulation promotes YY-1 protein, but not messenger RNA (mRNA) expression, via decreasing N6-methyladenosine methylation on YY-1 mRNA to suppress YTHDF2-medicated mRNA decay. Overall, this in-depth study shows that the activation of CD40L-CD40 signaling upregulates YY-1 protein expression in pulmonary adventitial fibroblasts, which results in increasing YY-1 and EZH2 binding to the IL-10 promoter region to enhance H3K27me3 modification, eventually leading to suppression of IL-10 transactivation. This study first uncovers the roles of YY-1 on CD40L-CD40 signaling-triggered inflammatory response in pulmonary adventitial fibroblasts.

Ras isoforms: signaling specificities in CD40 pathway.

BACKGROUND: Ras are small cellular GTPases which regulate diverse cellular processes. It has three isoforms: H-Ras, K-Ras, and N-Ras. Owing to the N-terminus (1-165 residues) sequence homology these isoforms were thought to be functionally redundant. However, only K-Ras-deficient mice but not H-Ras- and N-Ras-deficient mice show embryonic lethality. Similarly, mutations in a given Ras isoform are associated with a particular type of cancer. Moreover, we have previously reported that Ras isoforms perform unique functions in Leishmania major infection. Thus, Ras isoforms are implicated to have signaling and functional specificity but the mechanism remains to be elucidated. RESULT: Using CD40 as a model receptor, we showed that depending on the strength of signaling, specific Ras isoforms are activated. Weak CD40 signal activates N-Ras, whereas strong signal activates H-Ras and K-Ras. Additionally, we showed that suppression of N-Ras expression reduced CD40-induced extracellular signal-regulated kinase-1/2 (ERK-1/2) activation and Interleukin (IL)-10 production; whereas suppression of H-Ras or K-Ras reduced CD40-induced p38 mitogen-activated protein kinase (p38MAPK) activation and IL-12 production. Furthermore, we showed that Ras isoforms have activator (GEF) specificity as weak CD40 signal-activated N-Ras requires Sos-1/2 whereas strong CD40 signal-activated H-Ras/K-Ras requires Ras-GRP as the guanine-nucleotide exchange factor (GEF) inducing ERK-1/2- or p38MAPK-mediated IL-10 or IL-12 productions, respectively, in macrophages. Silencing of syk reduced CD40-induced N-Ras activation but silencing of lyn inhibited H-Ras and K-Ras activation. In CD40 signaling, Ras isoforms also showed effector specificity; while H-Ras and K-Ras showed specificity for phosphatidyl inositol-3 kinase activation at high dose of CD40 stimulation, N-Ras primarily associated with Raf-1 at low dose of CD40 stimulation. Moreover, fractal analysis showed that functional site surface roughness for H-Ras (SurfaceFD = 2.39) and K-Ras (SurfaceFD = 2.39) are similar but significantly different from N-Ras (SurfaceFD = 2.25). CONCLUSION: The activator and effector specificities of Ras isoforms in CD40 signaling indicated their differential involvement in CD40 pathway and in maintaining the reciprocity. Our observations reveal Ras-regulated signaling outcome and its potential for developing Ras isoform-targeted immunotherapy and prophylaxis.

Cytotoxic effect caspase activation dependent of a genetically engineered fusion protein with a CD154 peptide mimetic (OmpC-CD154p) on B-NHL cell lines is mediated by the inhibition of bcl-6 and YY1 through MAPK p38 activation.

The interaction between CD40, and its ligand, CD154, is essential for the development of humoral and cellular immune responses. The selective inhibition or activation of this pathway forms the basis for the development of new therapeutics against immunologically based diseases and malignancies. We are developing a gene fusion of Salmonella typhi OmpC protein expressing the CD154 Tyr140-Ser-149 amino acid strand. This OmpC-CD154 binds CD40 and activates B cells. In this study, we demonstrate that OmpC-CD154p treatment inhibits cell growth, proliferation and induced apoptosis in the B-NHL cell lines Raji and Ramos. The Bcl-2 family proteins were regulated and the Bcl-6 and YY1 oncoproteins were inhibited. p38 MAPK activation is an important mechanism underlying the effect on proliferation and apoptosis mediated by this fusion protein. This study establishes a basis for the possible use of fusion protein OmpC-CD154 as an alternative treatment for B-NHL.

Isorhamnetin, the active constituent of a Chinese herb Hippophae rhamnoides L, is a potent suppressor of dendritic-cell maturation and trafficking.

Dendritic cells (DCs) have been recognized as major targets of immunosuppressive therapies for their significant roles in connecting innate and adaptive immunity. Isorhamnetin (Iso), one of the most common flavonoid compounds extracted from the Chinese herb Hippophae rhamnoides L, has been proved to have anti-inflammatory, anticarcinogenic, and antioxidant activities in many chronic inflammatory conditions, but the effects of Iso on DCs have rarely been reported before. Here we investigated the functions and the mechanisms of Iso on bone marrow-derived DCs (BMDCs) including maturation, phagocytosis, and trafficking. Our data showed that Iso effectively inhibited the maturation of lipopolysaccharide (LPS)-treated BMDCs by down regulation of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß and IL-12p70, up regulation of IL-10, and depression of costimulatory molecules CD40, CD80, and CD86, while had no effects on phagocytosis. Furthermore, Iso inhibited the migration of LPS-treated BMDCs, which may be due to its inhibition on chemokine receptor 7 (CCR7) expression. These findings strongly suggest that Iso is a potent immunosuppressive agent by inhibiting DC activation and trafficking, and may be used to prevent or treat chronic inflammation, autoimmune diseases, and graft rejections.

In vitro inhibitory effects of thymol and carvacrol on dendritic cell activation and function.

CONTEXT: Thyme has been used in traditional medicine for medicinal purposes since ancient times. OBJECTIVE: The objective of this study was to investigate the effects of thymol and carvacrol as two major constituents of thyme on dendritic cells (DCs) maturation and T cell activation. MATERIALS AND METHODS: Splenic DCs were treated with non-cytotoxic concentrations of the components and then analyzed for MHC II, CD86, and CD40 expression by flow cytometry. The effects of compounds on mitogenic, as well as allogenic T cell responses in mixed lymphocyte culture (MLR) and the release of cytokines were investigated. RESULTS: At 0.1 µg/ml, reduced mean fluorescent intensity (MFI) of CD86 for thymol (80.3 ± 0.2% of untreated control) and CD40 for carvacrol (79.5 ± 0.14%) was observed (p < 0.001). Decreased mitogenic T cell proliferation by thymol [proliferation index (PI) from 0.93 ± 0.11 at 1 µg/ml to 0.42 ± 0.16 at 100 µg/ml (p < 0.01)] and carvacrol [PI from 1.08 ± 0.3 at 1 µg/ml to 0.28 ± 0.1 at 100 µg/ml (p < 0.001)] was seen. Ten micrograms/ml thymol (PI, 0.85 ± 0.04) and carvacrol (PI, 0.89 ± 0.03) inhibited allogenic T cell response (p < 0.05). Decreased IFN-γ level in MLR supernatant from 1441 ± 27.7 pg/ml in untreated cells to 944 ± 32.1 at 10 µg/ml of thymol and of carvacrol (886 ± 31.7 pg/ml) (p < 0.01) was found. IL-4 levels were decreased in the presence of both compounds (p < 0.01). CONCLUSION: These data showed the suppressive effects of thymol and carvacrol on DCs maturation and function, as well as T cell responses.

Nicotinic acid inhibits vascular inflammation via the SIRT1-dependent signaling pathway.

Nicotinic acid (NA) has recently been shown to inhibit inflammatory response in cardiovascular disease. Sirtuin1 (SIRT1), a NAD(+)-dependent class III histone deacetylase, participates in the regulation of cellular inflammation. We hypothesized that dietary supplementation of NA could attenuate vascular inflammation via modulation of SIRT1 pathway. New Zealand White rabbits received chow or chow supplemented with 0.6% (wt/wt) NA for 2 weeks. Acute vascular inflammation was induced in the animals by placing a non-occlusive silastic collar around the left common carotid artery. At 24 h after collar implantation, the collar-induced production of C-reactive protein and monocyte chemotactic protein-1 was significantly suppressed in the NA-supplemented animals. Meanwhile, NA also decreased the expression of cluster of differentiation 40 (CD40) and CD40 ligand, but up-regulated SIRT1 expression, both in rabbits and in lipopolysaccharide-stimulated endothelial cells. Moreover, knockdown of SIRT1 reversed the inhibitory effect of NA on CD40 expression. Further study revealed that NA also decreased the expression of CD40 partly through mammalian target of rapamycin. These results indicate that NA protects against vascular inflammation via the SIRT1/CD40-dependent signaling pathway.

Malaysian endophytic fungal extracts-induced anti-inflammation in Lipopolysaccharide-activated BV-2 microglia is associated with attenuation of NO production and, IL-6 and TNF-α expression.

BACKGROUND: Excessive production of inflammatory mediators such as nitric oxide (NO) and proinflammatory cytokines like tumour necrosis factor-alpha (TNF-α) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases. This study investigated the protective role of five endophytic extracts (HAB16R12, HAB16R13, HAB16R14, HAB16R18 and HAB8R24) against LPS-induced inflammatory events in vitro. These endophytic extracts were previously found to exhibit potent neuroprotective effect against LPS-challenged microglial cells. METHODS: The effects of these fungal endophytic extracts against nitric oxide (NO), CD40 phenotype and, pro- and anti-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated BV2 microglia cells were examined using commercially available assay kits, immunophenotyping and flow cytometry, respectively. RESULTS: Microglia pre-treated with the five endophytic extracts (0.1 mg/mL) reduced NO production without compromising cell viability. Whilst CD40 expression in LPS-stimulated microglia was not significantly different with or without the influence of endophytic extracts, expression of the proinflammatory cytokines, IL-6 and TNF-α in LPS-stimulated microglia was significantly (P < 0.05) inhibited by these endophytic extracts. CONCLUSIONS: The outcomes suggest that the neuroprotective effect of the fungal endophytic extracts is likely mediated through supression of neuroinflammation. To our knowledge, this is the first report of the effect of a fungal endophytic extract in controlling inflammation in BV2 microglia cells.

Anti-asthmatic effects of oxymatrine in a mouse model of allergic asthma through regulating CD40 signaling.

The aim of the study was to investigate the anti-asthmatic effects of oxymatrine (OXY) and the possible underlying mechanisms. The mouse asthma model was established by ovalbumin (OVA) intraperitoneal injection. A total of fifty mice were randomly assigned to five groups: control, OVA, OVA + dexamethasone (Dex, 2 mg · kg(-1)), and OVA + OXY (40 mg · kg(-1)), and OVA + OXY (80 mg · kg(-1)), respectively. Histological studies were conducted by the hematoxylin and eosin (HE) staining, the levels of interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13, and IgE were evaluated by enzyme-linked immunosorbent assay (ELISA), and the protein level of CD40 was analyzed by Western blotting. OXY inhibited OVA-induced increases in eosinophil count; the levels of IL-4, IL-5, IgE, and IL-13 were recovered. It also substantially inhibited OVA-induced eosinophilia in lung tissues and the expression of CD40 protein. These findings suggest that OXY may effectively ameliorate the progression of asthma and could be explored as a possible therapy for patients with allergic asthma.

[High Expression of co-stimulatory molecule CD40 in silicosis patients and intervention effect of yiqi huoxue decoction].

OBJECTIVE: To study whether co-stimulatory molecule CD40 of alveolar macrophage (AM) participated in the occurrence and development of silicosis, and to explore the intervention of Yiqi Huoxue Decoction (YHD) in the fibrosis of silicosis patients. METHODS: Totally 46 silicosis inpatients and outpatients were recruited and randomly assigned to the Western treatment group (A) and the Chinese medicine (CM) treatment group (B), 23 in each group. Patients in Group A received routine symptomatic treatment such as anti-inflammation, phlegm resolving, anti-spasm, and asthma relief, and so on. Patients in Group B additionally took YHD, one dose daily for 14 successive days. Besides, another 18 patients with chronic cough and sense of laryngeal foreign bodies were recruited as the normal control group, who had no obvious lesion confirmed by bronchofi6roscope and clinical diagnosis of the lung. They were treated by symptomatic supporting treatment. The alveolar lavage fluid was collected from all patients and isolated, and AM cells were cultured. The level of CD40 mRNA was detected by RT-PCR. The expression of CD40 protein was detected by Western blot. RESULTS: Compared with the normal control group, expression levels of CD40 mRNA and CD40 protein significantly increased in Group A (P < 0.01). Compared with Group A, expression levels of CD40 mRNA and CD40 protein significantly decreased in Group B (P < 0.01). CONCLUSIONS: Highly expressed co-stimulatory molecule CD40 of AM might participate in pulmonary fibrosis. YHD could hinder its roles, inhibit the progression of fibrosis, thereby playing an interventional role of treatment.

Irisflorentin modifies properties of mouse bone marrow-derived dendritic cells and reduces the allergic contact hypersensitivity responses.

Irisflorentin is an isoflavone component derived from the roots of Belamcanda chinensis (L.) DC. In traditional Chinese medicine, this herb has pharmacological properties to treat inflammatory disorders. Dendritic cells (DCs) are crucial modulators for the development of optimal T-cell immunity and maintenance of tolerance. Aberrant activation of DCs can induce harmful immune responses, and so agents that effectively improve DC properties have great clinical value. We herein investigated the effects of irisflorentin on lipopolysaccharide (LPS)-stimulated maturation of mouse bone marrow-derived DCs in vitro and in the contact hypersensitivity response (CHSR) in vivo. Our results demonstrated that treatment with up to 40 µM irisflorentin does not cause cellular toxicity. Irisflorentin significantly lessened the proinflammatory cytokine production (tumor necrosis factor-α, interleukin-6, and interleukin-12p70) by LPS-stimulated DCs. Irisflorentin also inhibited the expression of LPS-induced major histocompatibility complex class II and costimulatory molecules (CD40 and CD86) on LPS-stimulated DCs. In addition, irisflorentin diminished LPS-stimulated DC-elicited allogeneic T-cell proliferation. Furthermore, irisflorentin significantly interfered with LPS-induced activation of IκB kinase, c-Jun N-terminal kinase, and p38, as well as the nuclear translocation of NF-κB p65. Subsequently, treatment with irisflorentin obviously weakened 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity. These findings suggest new insights into the role of irisflorentin as an immunotherapeutic adjuvant through its capability to modulate the properties of DCs.

Obesity superimposed on aging magnifies inflammation and delays the resolving response after myocardial infarction.

Polyunsaturated fatty acid (PUFA) intake has increased over the last 100 yr, contributing to the current obesogenic environment. Obesity and aging are prominent risk factors for myocardial infarction (MI). How obesity interacts with aging to alter the post-MI response, however, is unclear. We tested the hypothesis that obesity in aging mice would impair the resolution of post-MI inflammation. PUFA diet (PUFA aging group) feeding to 12-mo-old C57BL/6J mice for 5 mo showed higher fat mass compared with standard lab chow (LC)-fed young (LC young group; 3-5 mo old) or aging alone control mice (LC aging group). LC young, LC aging, and PUFA aging mice were subjected to coronary artery ligation to induce MI. Despite similar infarct areas post-MI, plasma proteomic profiling revealed higher VCAM-1 in the PUFA aging group compared with LC young and LC aging groups, leading to increased neutrophil infiltration in the PUFA aging group (P<0.05). Macrophage inflammatory protein-1γ and CD40 were also increased at day 1, and myeloperoxidase remained elevated at day 5, an observation consistent with delayed wound healing in the PUFA aging group. Lipidomic analysis showed higher levels of arachidonic acid and 12(S)-hydroxyeicosatetraenoic acid at day 1 post-MI in the PUFA aging group compared with the LC aging group (all P<0.05), thereby mediating neutrophil extravasation in the PUFA aging group. The inflammation-resolving enzymes 5-lipoxygenase, cyclooxygenase-2, and heme oxyegnase-1 were altered to delay wound healing post-MI in the PUFA aging group compared with LC young and LC aging groups. PUFA aging magnifies the post-MI inflammatory response and impairs the healing response by stimulating prolonged neutrophil trafficking and proinflammatory lipid mediators.

Efficacy of Zhuling polyporus polysaccharide with BCG to inhibit bladder carcinoma.

There is growing interest in reducing Bacille Calmette-Guerin (BCG) side effects while keeping intact its therapeutic efficacy. In the present study, we evaluated the efficacy of Sclerotia of Polyporus umbellatus FRIES (Zhuling) and its main ingredient Polyporus Polysaccharide (PPS) to attenuate side effects of BCG therapy in vivo. The results show that bladder cancer development in model rats exhibited significantly reduced cancer invasiveness with Zhuling PPS combined with BCG. Flow cytometric (FCM) analysis showed expression of costimulatory molecules CD86, CD40, and TLR4/CD14 significantly increased with Zhuling PPS in combination with BCG. Similarly, immunohistochemical analysis revealed stronger CD86 and CD40 staining. Our findings show Zhuling PPS strongly reduced side effects and displayed synergistic effects during BCG instillation in rat bladder cancer models. The findings also suggest that the attenuation effect may result from direct activation of dendritic cell (DC) TLR4.

Immunomodulatory effects of Ziziphora tenuior L. extract on the dendritic cells.

BACKGROUND: Ziziphora tenuior L. (Kakuti in Persian) is used in traditional medicine for treatment of gastrointestinal disorders as carminative and analgesic plant. The other usages of this plant are included treatment of diarrhea and nausea. Therefore in the present study we evaluated the immunomodulatory effects of the ethanolic extract of this plant on the dendritic cells (DCs). RESULTS: Ziziphora tenuior L. extract significantly (p = 0.002) increased the level of surface expression of CD40 as an important co-stimulatory marker on DCs compared to the control. However this extract did not change CD86 and MHC-II molecules, so it could promote DCs phenotypic maturation. Treatment of DCs with the extract resulted in slightly increased of the production of (IL-12); however, this change was not significant. In addition, the ability of treated DCs to stimulate allogenic T cells proliferation and cytokines secretion was examined in the co-cuture of these cells with T cells in mixed lymphocyte reaction (MLR). Z. tenuior L. at the 100 µg/ml concentration inhibited the proliferation of allogenic T cells and also significantly (P < 0.001) increased the level of IL-10. Moreover, the extract at 10-100 µg/ml concentration caused slightly increase in IFN-γ production and decreased IL-4 cytokines but these changes were not significant. CONCLUSIONS: These findings indicated that Z. tenuior L. extract can modulate immune response by induction of CD40 expression on DCs and cytokine production; whereas it can inhibit T cell stimulating activity of DCs in high concentration. These findings possibly in part explain the traditional use of this plant in treatment of immune-mediated disorders. However future studies are needed.

Dietary wolfberry supplementation enhances the protective effect of flu vaccine against influenza challenge in aged mice.

Current vaccines for influenza do not fully protect the aged against influenza infection. Although wolfberry (goji berry) has been shown to improve immune response, including enhanced antibody production, after vaccination in the aged, it is not known if this effect would translate to better protection after influenza infection, nor is its underlying mechanism well understood. To address these issues, we conducted a study using a 2 × 2 design in which aged male mice (20-22 mo) were fed a control or a 5% wolfberry diet for 30 d, then immunized with an influenza vaccine or saline (control) on days 31 and 52 of the dietary intervention, and finally challenged with influenza A/Puerto Rico/8/34 virus. Mice fed wolfberry had higher influenza antibody titers and improved symptoms (less postinfection weight loss) compared with the mice treated by vaccine alone. Furthermore, an in vitro mechanistic study showed that wolfberry supplementation enhanced maturation and activity of antigen-presenting dendritic cells (DCs) in aged mice, as indicated by phenotypic change in expression of DC activation markers major histocompatibility complex class II, cluster of differentiation (CD) 40, CD80, and CD86, and functional change in DC production of cytokines interleukin-12 and tumor necrosis factor-α as well as DC endocytosis. Also, adoptive transfer of wolfberry-treated bone marrow DCs (loaded with ovalbumin(323-339)-peptide) promoted antigen-specific T cell proliferation as well as interleukin-4 and interferon-γ production in CD4(+) T cells. In summary, our data indicate that dietary wolfberry enhances the efficacy of influenza vaccination, resulting in better host protection to prevent subsequent influenza infection; this effect may be partly attributed to improved DC function.

Anti-inflammatory effects of danshen on human vascular endothelial cells in culture.

Inflammation plays a crucial role in the pathophysiology of atherosclerosis. Besides cytokines, chemokines and cell adhesion molecules, CD40 and P-selectin play important roles as key regulators of the inflammatory process in atherosclerosis. Danshen (DS) is commonly used in traditional Chinese medicine for therapy of cardiovascular diseases such as coronary artery disease. The aim of the present study was to evaluate the protective effects of DS with respect to possible anti-inflammatory effects. Human umbilical vein endothelial cells as well as platelets were incubated with an extract of DS or one of its major ingredients salvianolic acid B (Sal B), tanshinone IIA (Tansh) and protocatechuic acid (Protoc) under tumor necrosis factor (TNF)-α or ADP stimulation. Expression of CD40 and cellular adhesion molecules (VCAM-1/ICAM-1) were assessed via flow cytometry. Levels of interleukin (IL)-6, IL-8, monocyte-chemoattractant-protein (MCP)-1 as well as soluble VCAM1 and ICAM-1 in the supernatants were examined via luminex based analysis. Treatment with DS attenuated TNF-α induced expression of CD40. Furthermore, the expression of VCAM-1 and ICAM-1 as well as the release of soluble VCAM-1 and ICAM-1 were downregulated. In the cell supernatants we also observed a significant reduction of IL-6, IL8 and MCP-1. DS and its major ingredients, Sal B and Protoc, significantly inhibited TNF-induced expression and release of adhesion molecules, cytokines and chemokines as well as ADP-induced expression of platelet P-selectin. Because of the key roles of inflammatory mediators in the etiology of atherosclerosis, this work provides useful insight in understanding the pharmacological efficacy of Chinese herbal medicine.

Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Protective effect of magnesium lithospermate B against dextran sodiumsulfate induced ulcerative colitis in mice.

Anti-platelet drugs have been used to treat inflammatory bowel disease. In this study, we observed the therapeutic effects of magnesium lithospermate B, a main component of salvianolate, on colitis induced by dextran sodiumsulfate (DSS). Colitis was induced by 5% DSS oral administration in BALB/C male mice. Magnesium lithospermate B (60-240mg/kg) was given by subcutaneous injection for 2 weeks. Then, mice were sacrificed; serum and colon tissues were collected for biomarker assay, histological examination, immunohistochemical study and real-time quantitative polymerase chain reaction. DSS induced gross bleeding, inflammation, crypt damage and mucosal damage in colon. Treatment with magnesium lithospermate B could reduce colon inflammation induced by DSS. Magnesium lithospermate B could reverse the high CD40/CD40L expression and hypercoagulable state induced by DSS in colon. This study showed that magnesium lithospermate B could be used to treat colitis. The protective effects of magnesium lithospermate B may be due to its effects on CD40/CD40L expression and blood clotting status.

Omega-3 fatty acids enhance phagocytosis of Alzheimer's disease-related amyloid-ß42 by human microglia and decrease inflammatory markers.

The use of supplements with omega-3 (ω3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is widespread due to proposed beneficial effects on the nervous and cardiovascular systems. Many effects of ω3 FAs are believed to be caused by down-regulation and resolution of inflammation. Alzheimer's disease (AD) is associated with inflammation mediated by microglia and astrocytes, and ω3 FAs have been proposed as potential treatments for AD. The focus of the present study is on the effects of DHA and EPA on microglial phagocytosis of the AD pathogen amyloid-ß (Aß), on secreted and cellular markers of immune activity, and on production of brain-derived neurotrophic factor (BDNF). Human CHME3 microglial cells were exposed to DHA or EPA, with or without the presence of Aß42. Phagocytosis of Aß42 was analyzed by flow cytometry in conjunction with immunocytochemistry using antibodies to cellular proteins. Secreted proteins were analyzed by ELISA. Both DHA and EPA were found to stimulate microglial phagocytosis of Aß42. Phagocytosis of Aß42 was performed by microglia with a predominance of M2 markers. EPA increased the levels of BDNF in the culture medium. The levels of TNF-α were decreased by DHA. Both DHA and EPA decreased the pro-inflammatory M1 markers CD40 and CD86, and DHA had a stimulatory effect on the anti-inflammatory M2 marker CD206. DHA and EPA can be beneficial in AD by enhancing removal of Aß42, increasing neurotrophin production, decreasing pro-inflammatory cytokine production, and by inducing a shift in phenotype away from pro-inflammatory M1 activation.

Involvement of trypsin-digested silk peptides in the induction of RAW264.7 macrophage activation.

The activation of macrophages by trypsin-digested silk peptides was investigated by considering CD1 lb and CD40 expression in the RAW264.7 cell, a murine macrophage. Silk protein hydrolysates were digested with trypsin, following by centrifugal purification using the Centriprep 30k concentrator. Trypsin-digested total silk peptides and its centrifugal fractions were tested for macrophage activation in vitro. The functional peptide of fractionated silk peptides was examined by LC/MS/MS analysis. Trypsin-digested and fractionated silk peptides of more than 30 kDa induced an increase in the activation markers CD1 lb and CD40 in RAW264.7 cells. These results are supported by morphological changes reflecting an increase in the number of dendrites in activated cells. The fractionated silk peptides examined by LC/MS/MS contained partial peptides of Bombyx mori fibroin. These results suggest that the activation of RAW264.7 macrophages may be induced not by sericin-derived peptides but by fibroin-derived ones.