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ETHNOPHARMACOLOGICAL RELEVANCE: Coriolus versicolor (CV) has been used in traditional Chinese medicine for over 2000 years as a premium medicine for enhancing good health and longevity. The immunomodulatory and anti-cancer effects of polysaccharopeptides (PSP) from cultured CV have been extensively studied; however, the effect and the mechanism of action of other small molecules from CV remain unknown. AIM OF THE STUDY: we aim to examine the immunomodulatory and anti-cancer effects of the small molecules from CV (SMCV) and identify the active compounds that are responsible for the biological effects against glioblastoma multiforme cells. MATERIALS AND METHODS: The effects of SMCV/active compound on cytokine and MMP mRNA expressions and productions were assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. An active compound from SMCV was identified with a bioassay-guided fractionation scheme. The potential mode of action of the active compound was further investigated by identifying the cell signaling pathway. The protein expressions of phospho-ERK, phospho-JNK and phospho-p38 MAPKs were measured by Western Blotting. The anti-invasive effect of SMCV/bioactive compound against T98G, lung carcinoma (A549), and breast adenocarcinoma (MDA-MB-231) cells were determined using invasion assay. RESULTS: Our results showed that SMCV had strong immunomodulatory effect by suppressing LPS-induced TNF-α production, whereas increasing poly I:C-induced IFN-ß level in PBMac. SMCV not only possessed indirect anti-cancer effect by suppressing TNF-α-induced MMP-3 production in glioblastoma T98G cells, but also directly reduced the invasion ability of malignant cells including T98G, A549 and MDA-MB-231. Using bioassay-guided fractionation scheme, we isolated 9-KODE methyl ester (compound AM) that was responsible for the bioactivity of SMCV. This compound suppressed TNF-α-induced MMP-3 production in T98G cells and the suppression may be correlated with the inactivation of p38 mitogen-activated protein kinase (MAPK) pathway. Moreover, compound AM also directly reduced T98G cell invasion. CONCLUSION: Results of our present study provides scientific evidence that SMCV possesses immunomodulatory and anti-cancer effects. Its bioactive compound, compound AM, is a potential new drug candidate against the invasion and metastasis of glioblastoma cells.

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ETHNOPHARMACOLOGICAL RELEVANCE: Yinqiaosan is a classical traditional Chinese medicine formula, which has been used to treat respiratory diseases since ancient China. It consists of nine herbs and among them, Forsythia suspensa (Thunb.) Vahl fruit is one of the major herbal components. Despite the long history of Yinqiaosan, the active compounds and the mechanisms of action of this formula remain elusive. AIM OF THE STUDY: The present study aimed to examine the suppressive effect of Yinqiaosan on influenza virus and to identify the active components in the formula targeting influenza. MATERIALS AND METHODS: Anti-influenza virus effect of Yinqiaosan was assessed by tissue culture infective dose assay, and was also tested in an in vivo mouse model. Active compound from the formula was identified with a bioactivity-guided fractionation scheme. The potential mode of action of the compound was further investigated by identifying the host cell signaling pathways and viral protein production using in vitro cell culture models. RESULTS: Our results showed that forsythoside A from Forsythia suspensa (Thunb.) Vahl fruit, a major herbal component in Yinqiaosan, reduced the viral titers of different influenza virus subtypes in cell cultures and increased the survival rate of the mice in an in vivo influenza virus infection model. Further experiments on the mode of action of forsythoside A showed that it reduced the influenza M1 protein, which in turn intervened the budding process of the newly formed virions and eventually limited the virus spread. CONCLUSION: Results of our present study provides scientific evidence to support to the application of a traditional herbal formula. We also identify novel candidate compound for future drug development against influenza virus.

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Neuroinflammation and pro-inflammatory mediators play key roles in the pathogenesis of neurodegenerative diseases including stroke, which account for a significant burden of morbidity and mortality worldwide. Recently, the unsatisfactory pharmacotherapy and side effects of the drugs led to the development of alternative medicine for treating these diseases. Du Zhong (DZ), Eucommia ulmoides Oliver leaves, is a commonly used herb in the therapy of stroke in China. We hypothesize that the components from DZ inhibit neuroinflammation. In this study, DZ was extracted and the bioactive fractions with inhibitory effect on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in BV-2 microglial cells were further separated using chromatography. Two purified bioactive compounds, genipin (compound C) and 4-(1,2-dimethoxyethyl)benzene-1,2-diol (compound F), were isolated and identified after spectroscopic analysis. The results showed that they inhibited LPS-stimulated NO and tumor necrosis factor-alpha (TNF-α) production. Genipin exerted its anti-inflammatory effects through PI3K/Akt signaling pathway, whereas compound F inhibited phosphorylation of p38 mitogen-activated protein kinase (MAPK). In conclusion, genipin and compound F have potential for developing into new drugs for treating neurodegenerative diseases.

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Neurodegenerative diseases refer to the selective loss of neuronal systems in patients. The diseases cause high morbidity and mortality to approximately 22 million people worldwide and the number is expected to be tripled by 2050. Up to now, there is no effective prevention and treatment for the neurodegenerative diseases. Although some of the clinical therapies target at slowing down the progression of symptoms of the diseases, the general effectiveness of the drugs has been far from satisfactory. Traditional Chinese medicine becomes popular alternative remedies as it has been practiced clinically for more than thousands of years in China. As neurodegenerative diseases are mediated through different pathways, herbal decoction with multiple herbs is used as an effective therapeutic approach to work on multiple targets. Gastrodia and Uncaria Decoction, a popular TCM decoction, has been used to treat stroke in China. The decoction contains compounds including alkaloids, flavonoids, iridoids, carotenoids, and natural phenols, which have been found to possess anti-inflammatory, antioxidative, and antiapoptotic effects. In this review, we will summarize the recent publications of the pharmacological effects of these five groups of compounds. Understanding the mechanisms of action of these compounds may provide new treatment opportunities for the patients with neurodegenerative diseases.

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Interleukin 17A IL-17A is a crucial immunomodulator in various chronic immunological diseases including rheumatoid arthritis and inflammatory bowel disease. The cytokine has also been demonstrated to control the pathogenesis of the Mycobacterium tuberculosis by dysregulating production of cytokines and chemokines and promoting granuloma formation. Whether IL-17A regulates innate defence mechanisms of macrophages in response to mycobacterial infection remains to be elucidated. In the current report, we investigated the effects of IL-17A on modulating the intracellular survival of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in RAW264.7 murine macrophages. We observed that IL-17A pre-treatment for 24 hr was able to synergistically enhance BCG-induced nitric oxide (NO) production and inducible nitric oxide synthase expression in dose- and time-dependent manners. We further delineated the mechanisms involved in this synergistic reaction. IL-17A was found to specifically enhanced BCG-induced phosphorylation of Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. By using a specific JNK inhibitor (SP600125), we found that the production of NO in BCG-infected macrophages was significantly suppressed. Taken together, we confirmed the involvement of the JNK pathway in IL-17A-enhanced NO production in BCG-infected macrophages. We further demonstrated that IL-17A significantly enhanced the clearance of intracellular BCG by macrophages through an NO-dependent killing mechanism. In conclusion, our study revealed an anti-mycobacterial role of IL-17A through priming the macrophages to produce NO in response to mycobacterial infection.

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Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF)-alpha through p38 mitogen activated protein kinase (MAPK). However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1) and protein phosphatase type 2A (PP2A) in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac) infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

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BACKGROUND: Isolates of nonanthrax Bacillus species in clinical samples are frequently considered as contaminants. However, there were case reports describing Bacillus sepsis among infants, associated with high mortality and morbidity. METHODS: We performed a retrospective review of the clinical and epidemiological features of Bacillus bacteremia at our neonatal intensive care unit from January 2002 to December 2009. RESULTS: Bacillus bacteremia was considered to be clinically significant in 11 infants. The median gestational age was 30 weeks. All had either central catheters or peripherally inserted arterial lines in situ. The mean neutrophil and lymphocyte counts were 6.73 × 10(9)/L (0.78 to 12.56 × 10(9)/L) and 2.75 × 10(9)/L (0.82 to 6.15 × 10(9)/L), respectively. All 11 infants received intravenous vancomycin, with an average duration of 12.4 days. In general, the earlier the catheter was removed, the quicker the clearance of bacteremia was achieved. All infants survived and were discharged from the hospital. CONCLUSIONS: The growth of Bacillus species in blood cultures cannot simply be regarded as a contaminant. Hematologic parameters are frequently unremarkable at the disease onset. Increased vigilance, early diagnosis, and effective therapy in conjunction with prompt catheter removal are the keys to successful management of Bacillus bacteremia.

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Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disorder that causes permanent disability and mortality to approximately 1 to 100 people in the world. Patients with RA not only suffer from pain, stiffness, swelling, and loss of function in their joints, but also have a higher risk of cardiovascular disease and lymphoma. Typically prescribed medications, including pain-relieving drugs, nonsteroidal anti-inflammatory drugs (NSAID), and disease-modifying antirheumatic drugs, can help to relieve pain, reduce inflammation and slow the course of disease progression in RA patients. However, the general effectiveness of the drugs has been far from satisfactory. Other therapeutic modalities like TNF-alpha (TNF-α) inhibitors and interleukin-1 receptor antagonists targeting precise pathways within the immune system are expensive and may be associated with serious side effects. Recently, botanical medicines have become popular as alternative remedies as they are believed to be efficacious, safe and have over a thousand years experience in treating patients. In this review, we will summarize recent evidence for pharmacological effects of herbs including Black cohosh, Angelica sinensis, Licorice, Tripterygium wilfordii, Centella asiatica, and Urtica dioica. Scientific research has demonstrated that these herbs have strong anti-inflammatory and anti-arthritic effects. A wide range of phytochemicals including phenolic acids, phenylpropanoid ester, triterpene glycosides, phthalide, flavonoids, triterpenoid saponin, diterpene and triterpene have been isolated and demonstrated to be responsible for the biological effects of the herbs. Understanding the mechanisms of action of the herbs may provide new treatment opportunities for RA patients.

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We conducted a multicenter trial in Canada to assess the value of using trueness controls (TC) for rubella virus IgG and hepatitis B virus surface antibody (anti-HBs) serology to determine test performance across laboratories over time. TC were obtained from a single source with known international units. Seven laboratories using different test systems and kit lots included the TC in routine assay runs of the analytes. TC measurements of 1,095 rubella virus IgG and 1,195 anti-HBs runs were plotted on Levey-Jennings control charts for individual laboratories and analyzed using a multirule quality control (MQC) scheme as well as a single three-standard-deviation (3-SD) rule. All rubella virus IgG TC results were "in control" in only one of the seven laboratories. Among the rest, "out-of-control" results ranged from 5.6% to 10% with an outlier at 20.3% by MQC and from 1.1% to 5.6% with an outlier at 13.4% by the 3-SD rule. All anti-HBs TC results were "in control" in only two laboratories. Among the rest, "out-of-control" results ranged from 3.3% to 7.9% with an outlier at 19.8% by MQC and from 0% to 3.3% with an outlier at 10.5% by the 3-SD rule. In conclusion, through the continuous monitoring of assay performance using TC and quality control rules, our trial detected significant intra- and interlaboratory, test system, and kit lot variations for both analytes. In most cases the assay rejections could be attributable to the laboratories rather than to kit lots. This has implications for routine diagnostic screening and clinical practice guidelines and underscores the value of using an approach as described above for continuous quality improvement in result reporting and harmonization for these analytes.

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Dopamine toxicity is an ongoing controversy surrounding the use of levadopa (L-Dopa) in the therapy of Parkinson's disease. The initial objective of this study was to investigate the potential of neuroprotective botanicals such as Z-ligustilide in reducing the cytotoxicity of dopamine. We surprisingly found that Z-ligustilide potentiated dopamine toxicity in a dopaminergic cell specific manner. Using rat dopaminergic cell line PC12 as a model, we demonstrated that dopamine and Z-ligustilide in combination profoundly induced cell death, although these drugs alone, to a lesser extent, affected the cell viability in a concentration-dependent manner. The synergistic cytotoxicity of dopamine and Z-ligustilide is likely mediated via apoptosis, characterized by DNA fragmentation and chromatin shrinking after 12 h incubation. By measuring the intracellular reactive oxygen species (ROS) and reduced glutathione (GSH), Z-ligustilide and dopamine in combination dramatically enhanced the ROS formation and further depleted reduced GSH, whereas these drugs alone showed much less activity. Importantly, the synergistic cytotoxicity of dopamine and Z-ligustilide could be largely prevented by thiol-containing antioxidant N-acetylcysteine and GSH other than vitamin C and Trolox. Since the cytotoxicity of Z-ligustilide was not reported previously, the results of this study should raise public concerns over the potential risk associated with the combined use of herbal medicines containing Z-ligustilide with L-Dopa in the therapy of Parkinson's disease.

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c-Myc (Myc) is a well known transcription factor that regulates many essential cellular processes; however, its role in modulating immunity is not known. Here, we showed different species of mycobacteria can induce Myc expression via ERK1/2 and JNK activation. Unexpectedly, the induced Myc is localized in the cytoplasm but not in the nucleus. This induced Myc expression is associated with the induction of TNF-α and IL-6 and with the suppression of intracellular mycobacterial growth. To delineate the underlying mechanisms, we demonstrated that Myc enhances IRAK1 degradation, leading to specific activations of ERK1/2 and p38 MAPK but not Akt, and reduces IκBα protein recovery upon degradation. Hence, our findings may provide insights into a potential role for Myc in regulating the antimicrobial responses.

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Leukotriene B4-12-hydroxydehydrogenase (LTB4DH) is characterized as a chemopreventive and tumor suppressor gene. The aim of this study was to investigate the pharmaco-logical induction of LTB4DH and potential anticancer activity. Using HepG2 cells as a cellular detector, we successfully isolated the active compounds from the herbs Radix Astragali and Radix Paeoniae Rubra through a bioactivity-guided fractionation procedure. Using various analytical techniques including electronic spray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR), gallic acid (GA) was identified as the active compound from Radix Paeoniae Rubra whereas the active compound from Radix Astragali, designated as RA-C, was also purified to the extent that it is now suitable for further identifi-cation. We found that the active compounds from these two different herbs synergistically induced LTB4DH expression in a dose- and time-dependent manner. A key finding was that commercial GA in combination with purified RA-C attenuated the focus formation and anchorage-independent growth, two indexes of in vitro oncogenic transformation, of HepG2 cells via the induction of LTB4DH expression. Moreover, the combination of GA and purified RA-C significantly induced G2/M cell cycle arrest in HepG2 cells. Our results demon-strated for the first time that GA and purified RA-C suppress the in vitro oncogenic transformation of HepG2 cells via the induction of LTB4DH expression. Importantly, pharmaco-logical induction of LTB4DH represents a potential alternative strategy for the therapy of hepatocellular carcinoma.

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Panax ginseng is one of the most commonly used Chinese medicines in China, Asia and Western countries. The beneficial effects of ginseng have been attributed to the biological activities of its constituents, the ginsenosides. In this review, we summarize recent publications on the anti-inflammatory effects of ginseng extracts and ginsenosides on cellular responses triggered by different inducers including endotoxin, tumor necrosis factor-alpha (TNF-α), interferon-gamma and other stimuli. Proinflammatory cytokines, chemokines, adhesion molecules and mediators of inflammation including inducible nitric oxide synthase, cyclooxygenase-2 and nitric oxide orchestrate the inflammatory response. Ginseng extracts and ginsenosides including Rb1, Rd, Rg1, Rg3, Rh1, Rh2, Rh3 and Rp1 have been reported to have anti-inflammatory properties in different studies related to inflammation. Ginsenosides inhibit different inducers-activated signaling protein kinases and transcription factor nuclear factor-kappaB leading to decreases in the production of cytokines and mediators of inflammation. The therapeutic potential of ginseng on TNF-α-mediated inflammatory diseases is also discussed. Taken together, this summary provides evidences for the anti-inflammatory effects of ginseng extracts and ginsenosides as well as the underlying mechanisms of their effects on inflammatory diseases.

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The pathogenesis of Mtb depends in part on cytokine cross-regulation between macrophages and T cells in host immunity. Th17 cells produce IL-17A to induce granuloma formation and to restrict mycobacterial dissemination. IL-17A also mediates cytokine responses induced by proinflammatory cytokines such as TNF-α. Our previous results showed that BCG induces IL-6, IL-10, and TNF-α via activity of protein kinases, including dsRNA-activated serine/threonine protein kinase and glycogen synthase kinase-3 in primary human monocytes. Therefore, we investigated whether IL-17A, upon its induction by BCG, plays an additional role to aid the production of downstream proinflammatory cytokines in macrophages. Here, we showed that IL-17A enhanced IL-6 mRNA and protein levels inducible by BCG in a time- and dose-dependent manner, whereas it had no effect on IL-10 and TNF-α production. We also demonstrated that IL-17A activated the phosphorylation of ERK1/2 triggered by BCG. With the use of a specific chemical inhibitor of a MAPK/ERK-activating kinase (MEK1/2), we confirmed the correlation between the enhanced ERK1/2 activation and augmented IL-6 production. Additionally, we revealed that IL-17A acts in concert with BCG-induced TNF-α to enhance the level of IL-6 synthesis. Taken together, our results suggest a significant role of IL-17A to serve as a modulator of cytokine expression in innate immune response during mycobacterial infection.

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BACKGROUND: Upon initial infection with mycobacteria, macrophages secrete multiple cytokines and chemokines, including interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α), to mediate host immune responses against the pathogen. Mycobacteria also induce the production of IL-10 via PKR activation in primary human monocytes and macrophages. As an anti-inflammatory cytokine, over-expression of IL-10 may contribute to mycobacterial evasion of the host immunity. Radix Paeoniae Rubra (RPR, Chishao), a Chinese medicinal herb with potentials of anti-inflammatory, hepatoprotective and neuroprotective effects, is used to treat tuberculosis. This study investigates the immunoregulatory effects of RPR on primary human blood macrophages (PBMac) during mycobacterial infection. METHODS: The interaction of Bacillus Calmette-Guerin (BCG) with PBMac was used as an experimental model. A series of procedures involving solvent extraction and fractionation were used to isolate bioactive constituents in RPR. RPR-EA-S1, a fraction with potent immunoregulatory effects was obtained with a bioactivity guided fractionation scheme. PBMac were treated with crude RPR extracts or RPR-EA-S1 before BCG stimulation. The expression levels of IL-6, IL-8, IL-10 and TNF-α were measured by qPCR and ELISA. Western blotting was used to determine the effects of RPR-EA-S1 on signaling kinases and transcriptional factors in the BCG-activated PBMac. RESULTS: In BCG-stimulated macrophages, crude RPR extracts and fraction RPR-EA-S1 specifically inhibited IL-10 production while enhanced IL-8 expression at both mRNA and protein levels without affecting the expressions of IL-6 and TNF-α. Inhibition of BCG-induced IL-10 expression by RPR-EA-S1 occurred in a dose- and time-dependent manner. RPR-EA-S1 did not affect the phosphorylation of cellular protein kinases including MAPK, Akt and GSK3ß. Instead, it suppressed the degradation of IκBα in the cytoplasm and inhibited the translocation of transcription factor NF-κB1 p50 to the nucleus. CONCLUSION: RPR crude extracts and its fraction RPR-EA-S1 inhibited anti-inflammatory cytokine IL-10 and enhanced pro-inflammatory chemokine IL-8 expression in BCG-activated PBMac. The inhibitory effects of RPR-EA-S1 on IL-10 expression in BCG-activated PBMac may be due to the reduced nuclear translocation of NF-κB1 p50.

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Stroke is the third most common cause of death worldwide. Recent findings showed that the severity of cerebrovascular diseases including ischemic stroke correlates with inflammation mediated responses in the neural cells. During ischemia, inflammatory mediators including tumor necrosis factor-alpha (TNF-α) and nitric oxide are produced by microglia, which play a central role in the pathogenesis of the disease. Ligusticum chuanxiong (LCX) is a commonly used traditional Chinese medicine (TCM) for empiric treatment of cerebrovascular and cardiovascular diseases for many centuries. By applying a bioactivity-guided fractionation scheme, two compounds with inhibition on neuroinflammation were isolated from LCX. Using chromatographic and spectrometric methods, they were identified to be senkyunolide A and Z-ligustilide. They could inhibit the production of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV-2 microglial cells and human peripheral blood monocyte derived macrophages. In addition, both compounds protected Neuro-2a cells from neuroinflammatory toxicity induced by the conditioned culture media produced by LPS-stimulated BV-2 cells. The underlying mechanisms of action of senkyunolide A were further delineated. Its inhibitory effects were shown to be independent of the phosphorylation of mitogen-activated protein kinases (MAPK) and translocation of nuclear factor kappa B (NF-κB). However, senkyunolide A could increase the degradation of TNF-α mRNA and reduce its half life by 43%. In conclusion, bioactivity-guided fractionation is an effective way of isolating bioactive compounds from medicinal herbs. In addition, senkyunolide A and Z-ligustilide isolated from LCX may be considered as potential complementary drug candidates for treating inflammatory processes associated with cerebrovascular diseases.

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OBJECTIVE AND DESIGN: HIV-1 transactivator protein, Tat, has been identified as an activator of HIV-1 replication. It also dysregulates cytokine production and apoptosis in T-cells. Of the various cell death processes, autophagy is a self-digestion and degradation mechanism that recycles the contents of the cytosol, including macromolecules and cellular organelles, resulting in self-repair and conservation for survival. Recent reports demonstrated that autophagosomes can be activated by interferon-γ (IFN-γ) to participate in immune defence by processing foreign antigens for the recognition and killing of intracellular pathogens. As we previously showed that HIV-1 Tat perturbs IFN-γ signaling through the suppression of STAT1 phosphorylation and consequently inhibits major histocompatibility complex class-II antigen expression, we postulate that Tat plays a role in regulating autophagy. METHODS: The role of STAT1 in IFN-γ-induced autophagy in primary human blood macrophages was examined using a small molecule inhibitor or siRNA specific for STAT1. The effect of HIV-1 Tat on autophagy was investigated by pretreating the macrophages with HIV-1 Tat and followed by IFN-γ stimulation. The expressions of autophagy-associated genes and their effects on engulfing mycobacteria were examined. RESULTS: The activation of STAT1 resulted in IFN-γ-induced LC3B protein expression and autophagosome formation. As postulated, HIV-1 Tat protein suppressed IFN-γ-induced autophagy processes, including LC3B expression. Additionally, HIV-1 Tat restricted the capturing of mycobacteria by autophagosomes. CONCLUSION: HIV-1 Tat suppressed the induction of autophagy-associated genes and inhibited the formation of autophagosomes. Perturbation of such cellular processes by HIV-1 would impair the effective containment of invading pathogens, thereby providing a favorable environment for opportunistic microbes in HIV-infected individuals.

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Historically, influenza pandemics have arisen from avian influenza viruses. Avian influenza viruses H5N1 and H9N2 are potential pandemic candidates. Infection of humans with the highly pathogenic avian influenza H5N1 virus is associated with a mortality in excess of 60%, which has been attributed to dysregulation of the cytokine system. Human macrophages and epithelial cells infected with some genotypes of H5N1 and H9N2 viruses express markedly elevated cytokine and chemokine levels when compared with seasonal influenza A subtype H1N1 virus. The mechanisms underlying this cytokine and chemokine hyperinduction are not fully elucidated. In the present study, we demonstrate that autophagy, a tightly regulated homeostatic process for self-digestion of unwanted cellular subcomponents, plays a role in cytokine induction. Autophagy is induced to a greater extent by H9N2/G1, in association with cytokine hyperinduction, compared with H1N1 and the novel pandemic swine-origin influenza A/H1N1 viruses. Using 3-methyladenine to inhibit autophagy and small interfering RNA to silence the autophagy gene, Atg5, we further show that autophagic responses play a role in influenza virus-induced CXCL10 and interferon-alpha expression in primary human blood macrophages. Our results provide new insights into the pathogenic mechanisms of avian influenza viruses.

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IL-10, a potent anti-inflammatory cytokine, activates its primary mediator STAT3 to exert inhibitory effects on activated immune response. It has been reported that IFN-gamma signaling can be suppressed by IL-10, which deactivates macrophages and suppresses cell-mediated antigen presentation. Cathepsin S, a cysteine protease, plays a significant role in the antigen processing. We hypothesize that the IL-10-induced and STAT3-mediated signaling pathway interferes with IFN-gamma-induced immune responses in primary human blood macrophages. Here, we investigated whether IL-10 perturbs MHC-II levels via its effect on cathepsin S expression in antigen processing. We showed that the expression of cathepsin S and MHC-II, inducible by IFN-gamma, was down-regulated in the presence of IL-10. Additionally, we revealed that the inhibitory effect of IL-10 was demonstrated to be independent of the classical IFN-gamma-induced JAK2/STAT1 signaling cascade or the NF-kappaB pathway. Following STAT3 suppression with specific siRNA, the expression of IFN-gamma-induced surface MHC-II antigens and cathepsin S levels was restored, even in the presence of IL-10. Taken together, our results demonstrated that the immunosuppressive effects of IL-10-STAT3 on MHC-II antigen presentation may occur via the inhibition of cathepsin S expression.

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