Search

Developing highly effective HIV latency-reversing agent is an inportmant approach for the treatment of AIDS via the "shock and kill" of latent HIV. In this study, two unreported modified daphnane-type diterpenes (chamaedaphnelide A and epi-chamaedaphnelide A) and one unreported tigliane-type diterpene (chamaedaphnelide B), along with four known daphnane-type diterpenes and one known tigliane-type diterpene were obtained from the leaves of Wikstroemia chamaedaphne. Chamaedaphnelide A and epi-chamaedaphnelide A represents the first A ring cleavage daphnane-type backbone. Chamaedaphnelide A, epi-chamaedaphnelide A, chamaedaphnelide B, and 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate showed HIV latency-reversing activity, especially chamaedaphnelide B and 6α,7α-epoxy-5β-hydroxy-12-deoxyphorbol-13-decanoate displayed equally potential to positive drugs prostratin with reversing latent HIV on more than 100-fold compared to unstimulated cells. Furthermore, the activation of STAT1 was involved in the HIV latency-reversing activity of these diterpenes, firstly demonstrating that daphnane- and tigliane-type diterpenes can rapidly activate STAT1 activity. Indeed, these results also supported that activating STAT1 activity is a pathway for reversing latent HIV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Schisandrin B (Sch B) is the major active ingredient of the traditional Chinese medicine Schisandra chinensis and has antitumor activity, anti-inflammatory activity. CD4 + Th subsets orchestrate immune responses to plenty of pathogen infections and participate in the pathogenesis of many immune-related diseases. However, little is known about the relationship between Sch B and T cell differentiation. Here, we showed that Sch B might participate in T cell receptor signaling pathway by using the TCMIO database. Importantly, Sch B promoted T H 1 cell differentiation. Furthermore, Sch B did not affect T H 2 cell and Treg differentiation. Mechanismly, Sch B increased the level of IFN-γ of CD4 + T cells by upregulating the phosphorylation of STAT1 protein. Then, STAT1 promoted T-bet expression in CD4 + T cells. In conclusion, Sch B modulates the differentiation of naïve CD4 + T cells into T H 1 subset by STAT1/T-bet signaling, which may have the potential for the treatment of T cell-mediated-immune diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objectives: SLE is characterized by two pathogenic key signatures, type I IFN and B-cell abnormalities. How these signatures are interrelated is not known. Type I-II IFN trigger activation of Janus kinase (JAK) - signal transducer and activator of transcription (STAT). JAK-STAT inhibition is an attractive therapeutic possibility for SLE. We assess STAT1 and STAT3 expression and phosphorylation at baseline and after IFN type I and II stimulation in B-cell subpopulations of SLE patients compared with other autoimmune diseases and healthy controls (HD) and related it to disease activity., Methods: Expression of STAT1, pSTAT1, STAT3 and pSTAT3 in B and T cells of 21 HD, 10 rheumatoid arthritis (RA), seven primary Sjögren's (pSS) and 22 SLE patients was analysed by flow cytometry. STAT1 and STAT3 expression and phosphorylation in PBMCs (peripheral blood mononuclear cells) of SLE patients and HD after IFNα and IFNγ incubation were further investigated., Results: SLE patients showed substantially higher STAT1 but not pSTAT1 in B- and T-cell subsets. Increased STAT1 expression in B-cell subsets correlated significantly with SLEDAI and Siglec-1 on monocytes, a type I IFN marker. STAT1 activation in plasmablasts was IFNα dependent while monocytes exhibited dependence on IFNγ., Conclusion: Enhanced expression of STAT1 by B-cell candidates as a key node of two immunopathogenic signatures (type I IFN and B-cells) related to important immunopathogenic pathways and lupus activity. We show that STAT1 is activated upon IFNα exposure in SLE plasmablasts. Thus, Jak inhibitors, targeting JAK-STAT pathways, hold a promise to block STAT1 expression and control plasmablast induction in SLE., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Liver fibrosis constitutes a major health problem worldwide due to its rapidly increasing prevalence and the lack of specific and effective treatments. Growing evidence suggests that signalling through cytokine-activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways regulates liver fibrosis and regeneration. Rilpivirine (RPV) is a widely used anti-HIV drug not reported to produce hepatotoxicity. We aimed to describe the potential hepatoprotective effects of RPV in different models of chronic liver injury, focusing on JAK-STAT signalling regulation., Design: The effects of RPV on hepatic steatosis, inflammation and fibrogenesis were studied in a nutritional mouse model of non-alcoholic fatty liver disease, carbon tetrachloride-induced fibrosis and bile duct ligation-induced fibrosis. Primary human hepatic stellate cells (hHSC) and human cell lines LX-2 and Hep3B were used to investigate the underlying molecular mechanisms., Results: RPV exerted a clear anti-inflammatory and antifibrotic effect in all the in vivo models of liver injury employed, and enhanced STAT3-dependent proliferation in hepatocytes and apoptosis in HSC through selective STAT1 activation. These results were reproduced in vitro; RPV undermined STAT3 activation and triggered STAT1-mediated pathways and apoptosis in HSC. Interestingly, this selective pro-apoptotic effect completely disappeared when STAT1 was silenced. Conditioned medium experiments showed that HSC apoptosis activated STAT3 in hepatocytes in an interleukin-6-dependent mechanism., Conclusion: RPV ameliorates liver fibrosis through selective STAT1-dependent induction of apoptosis in HSC, which exert paracrinal effects in hepatocytes, thus promoting liver regeneration. RPV's actions may represent an effective strategy to treat chronic liver diseases of different aetiologies and help identify novel therapeutic targets., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients exhibit a gene expression program (interferon [IFN] signature) that is attributed to overproduction of type I IFNs by plasmacytoid dendritic cells. Type I IFNs have been thought to play a role in the pathogenesis of SLE. This study was undertaken to examine an unexpected influence of monocyte/macrophages on the IFN signature., Methods: Proinflammatory (classic) and antiinflammatory (nonclassic) monocyte/macrophages were sorted from mice and analyzed by RNA sequencing and quantitative polymerase chain reaction (qPCR). Type I IFN-α/β/ω receptor (IFNAR-1) expression was determined by qPCR and flow cytometry. Macrophages were stimulated in vitro with IFNα, and pSTAT1was measured., Results: Transcriptional profiling of peritoneal macrophages from mice with pristane-induced SLE unexpectedly indicated a strong IFN signature in classic, but not nonclassic, monocyte/macrophages exposed to the same type I IFN concentrations. Ifnar1 messenger RNA and IFNAR surface staining were higher in classic monocyte/macrophages versus nonclassic monocyte/macrophages (P < 0.0001 and P < 0.05, respectively, by Student's t-test). Nonclassic monocyte/macrophages were also relatively insensitive to IFNα-driven STAT1 phosphorylation. Humans exhibited a similar pattern: higher IFNAR expression (P < 0.0001 by Student's t-test) and IFNα-stimulated gene expression (P < 0.01 by paired Wilcoxon's rank sum test) in classic monocyte/macrophages and lower levels in nonclassic monocyte/macrophages., Conclusion: This study revealed that the relative abundance of different monocyte/macrophage subsets helps determine the magnitude of the IFN signature. Responsiveness to IFNα signaling reflects differences in IFNAR expression in classic (high IFNAR) compared to nonclassic (low IFNAR) monocyte/macrophages. Thus, the IFN signature depends on both type I IFN production and the responsiveness of monocyte/macrophages to IFNAR signaling., (© 2019, American College of Rheumatology.)

Compelling evidence indicates that epigenetic regulations orchestrate dynamic macrophage polarization. N 6 -methyladenosine (m 6 A) methylation is the most abundant epigenetic modification of mammalian mRNA, but its role in macrophage polarization is still completely unknown. Here, we show that the m 6 A-catalytic enzyme methyltransferase like 3 (METTL3) is specifically upregulated following the M1 polarization of mouse macrophages. Furthermore, METTL3 knockdown through siRNA transfection markedly inhibited M1, but enhanced M2, macrophage polarization. Conversely, its overexpression via plasmid transfection greatly facilitated M1, but attenuated M2, macrophage polarization. Further methylated RNA immunoprecipitation and in vitro m 6 A methylation assays suggested that METTL3 directly methylates mRNA encoding signal transducer and activator of transcription 1 (STAT1), a master transcription factor controlling M1 macrophage polarization, at its coding sequence and 3'-untranslated regions. In addition, METTL3-mediated STAT1 mRNA methylation significantly increased mRNA stability and subsequently upregulated STAT1 expression. In conclusion, METTL3 drives M1 macrophage polarization by directly methylating STAT1 mRNA, potentially serving as an anti-inflammatory target.

Background: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immune-evasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-γ modulation of PD-L1/PD-L2 in MET-amplified tumours., Methods: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-γ were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-γ stimulation, and after anti-MET therapy., Results: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-γ treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/ STAT1, signal transducers downstream of the Interferon-γ receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-γ induced PD-L1 expression., Conclusions: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-γ. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

Objectives: Breast cancer is the most commonly diagnosed cancer, and it is a leading cause of cancer-related deaths in females worldwide. Triple-negative breast cancer (TNBC) constitutes 15% of breast cancer and shows distinct metastasis profiles with poor prognosis. Strong PD-L1 expression has been observed in some tumors, supporting their escape from immune surveillance. Targeting PD-L1 could be a promising therapeutic approach in breast cancer patients. We investigated potential molecular mechanisms for constitutive expression of PD-L1 by inhibiting upstream STAT1 and STAT3 signals., Methods: PD-L1 expression in three breast cancer cell lines was measured using quantitative PCR and western blotting. Activation of STAT1 and STAT3 was blocked using pharmacological inhibitors and siRNA. The mechanism underlying the constitutive expression of PD-L1 was investigated using ChIP and co-immunoprecipitation assays., Results: We found that individual inhibition of STAT1 and STAT3 activation partially downregulated PD-L1, while combined inhibition completely downregulated PD-L1 expression. Moreover, our results suggest that pSTAT1-pSTAT3 dimerize in cytosol and translocate to the nucleus, where they bind to PD-L1 promoter and induce PD-L1 expression., Conclusion: These findings provide a rationale for combined targeting of STAT1 and STAT3 for the development of immune-based cancer therapies for down regulation of PD-L1 expression.

Background/aims: Grape seed proanthocyanidin extract (GSPE) has been reported to have a beneficial effect on regulating inf lammation. However, the anti-inflammatory mechanism of GSPE remains unclear. The aim of this study was to verify the influence of GSPE on the Toll-like receptor 4 (TLR4)-mediated signaling pathway in the regulation of murine autoimmune arthritis., Methods: Collagen-induced arthritis (CIA) was induced in dilute brown non-agouti (DBA)/1J mice. The mice were treated with GSPE (0 or 100 mg/kg) intraperitoneally. The severity of arthritis was assessed clinically, biochemically, and histologically. Immunostaining for TLR4 was performed. The expressions of TLR4 and downstream signaling molecules were analyzed by Western blot. The effect of GSPE on lipopolysaccharide (LPS)-induced TLR4 activation was also evaluated using RAW264.7 cells and fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis and from those with osteoarthritis., Results: GSPE attenuated the clinical severity of arthritis and decreased histological damage. GSPE treatment reduced the number of TLR4-stained cells in the synovium of mice with CIA. GSPE also downregulated the expression of TLR4, myeloid differentiation factor 88 (MyD88) and phosphorylated IκBα synovial protein in CIA mice. Concurrently, GSPE inhibited the nuclear translocation of nuclear factor-κB (NF-κB) subunits (p65 and p50). LPS-induced TLR4 activation was suppressed by GSPE in human FLS as well as in murine macrophages in vitro ., Conclusions: Our results demonstrated that GSPE ameliorated CIA by regulating the TLR4-MyD88-NF-κB signaling pathway.

Background: In humans, interferon-α treatment for chronic viral hepatitis is a well-recognized clinical model for inflammation-induced depression, but the molecular mechanisms underlying these effects are not clear. Following peripheral administration in rodents, interferon-α induces signal transducer and activator of transcription-1 (STAT1) within the hippocampus and disrupts hippocampal neurogenesis., Methods: We used the human hippocampal progenitor cell line HPC0A07/03C to evaluate the effects of 2 concentrations of interferon-α, similar to those observed in human serum during its therapeutic use (500 pg/mL and 5000 pg/mL), on neurogenesis and apoptosis., Results: Both concentrations of interferon-α decreased hippocampal neurogenesis, with the high concentration also increasing apoptosis. Moreover, interferon-α increased the expression of interferon-stimulated gene 15 (ISG15), ubiquitin-specific peptidase 18 (USP18), and interleukin-6 (IL-6) via activation of STAT1. Like interferon-α, co-treatment with a combination of ISG15, USP18, and IL-6 was able to reduce neurogenesis and enhance apoptosis via further downstream activation of STAT1. Further experiments showed that ISG15 and USP18 mediated the interferon-α-induced reduction in neurogenesis (potentially through upregulation of the ISGylation-related proteins UBA7, UBE2L6, and HERC5), while IL-6 mediated the interferon-α-induced increase in apoptosis (potentially through downregulation of aquaporin 4). Using transcriptomic analyses, we showed that interferon-α regulated pathways involved in oxidative stress and immune response (e.g., Nuclear Factor (erythroid-derived 2)-like 2 [Nrf2] and interferon regulatory factor [IRF] signaling pathway), neuronal formation (e.g., CAMP response element-binding protein [CREB] signaling), and cell death regulation (e.g., tumor protein(p)53 signaling)., Conclusions: We identify novel molecular mechanisms mediating the effects of interferon-α on the human hippocampus potentially involved in inflammation-induced neuropsychiatric symptoms., (© The Author 2017. Published by Oxford University Press on behalf of CINP.)

IL-7 regulates homeostatic mechanisms that maintain the overall size of the T cell pool throughout life. We show that, under steady-state conditions, IL-7 signaling is principally mediated by activation of signal transducers and activators of transcription 5 (STAT5). In contrast, under lymphopenic conditions, there is a modulation of STAT1 expression resulting in an IL-7-dependent STAT1 and STAT5 activation. Consequently, the IL-7-induced transcriptome is altered with enrichment of IFN-stimulated genes (ISGs). Moreover, STAT1 overexpression was associated with reduced survival in CD4+ T cells undergoing lymphopenia-induced proliferation (LIP). We propose a model in which T cells undergoing LIP upregulate STAT1 protein, "switching on" an alternate IL-7-dependent program. This mechanism could be a physiological process to regulate the expansion and size of the CD4+ T cell pool. During HIV infection, the virus could exploit this pathway, leading to the homeostatic dysregulation of the T cell pools observed in these patients.

Objective: Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING., Methods: PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNβ reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression., Results: Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNβ promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade., Conclusion: These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies., (© 2017, American College of Rheumatology.)

γ-Aminobutyric acid (GABA) is a major amino acid neurotransmitter in the vertebrate brain. To provide detailed information on the distribution of the GABA in zebrafish (Danio rerio), neurons were labeled with mCherry driven by the glutamic acid decarboxylase 67 (gad67) promoter. In the transgenic line Tg(gad67:mCherry), mCherry-positive gad67 cell bodies were predominantly localized to the olfactory bulb, pallial zones, subpallium zones, parvocellular preoptic nucleus, periventricular gray zone of optic tectum, torus semicircularis, posterior tuberculum, medial longitudinal fascicle, caudal zone of periventricular hypothalamus, and oculomotor nucleus. mCherry-positive fibers were widely distributed in the olfactory bulbs, subpallium, thalamus, ventral hypothalamic zone, tectum opticum, mesencephalon, and rhombencephalon. mCherry-positive neurons were also observed in the retina and the spinal cord. The anatomical relationships between GABAergic and gonadotrophin-releasing hormone 3 (GnRH3) neurons were investigated by crossing Tg(gad67:mCherry) fish with the previously established Tg(gnrh3:EGFP) transgenic line. GnRH3 cell bodies and fibers were contacted by GABAergic fibers directly in the ventral telencephalon and anterior tuberal nucleus. A subpopulation of GnRH3 neurons in the ventral telencephalic area was also labeled with mCherry, so some GnRH3 neurons are also GABAergic. GABAB receptor agonist (baclofen) and antagonist (CGP55845) treatments indicated that GABAB receptor signaling inhibited gnrh3 expression in larval fish but was stimulatory in adult fish. The expression of pituitary lhβ and fshβ was stimulated by intraperitoneal injection of baclofen in adult fish. We conclude that GABA via GABAB receptors regulates GnRH3 neurons in a developmentally dependent manner in zebrafish., (Copyright © 2017 Endocrine Society.)

Background: Emodin (3-methyl-1, 6, 8-trihydroxyanthraquinone) is a compound which can be found in Polygoni Multiflori Radix (PMR). PMR is the root of Polygonum multiflorum . PMR is used to treat dizziness, spermatorrhea, sores, and scrofula as well as chronic malaria traditionally in China and Korea. The anti-tumor property of emodin was already reported. However, anti-viral activity of emodin on macrophages are not fully reported., Materials and Methods: Effects of emodin on RAW 264.7 mouse macrophages induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA, were evaluated., Results: Emodin restored the cell viability in poly I: C-induced RAW 264.7 at concentrations of up to 50 μM. Emodin significantly inhibited the production of nitric oxide, IL-1α, IL-Ιβ, IL-6, GM-CSF, G-CSF, M-CSF, MCP-1, MIP-1a, MIP-Ιβ, MIP-2, RANTES, and IP-10 as well as calcium release and mRNA expression of signal transducer and activated transcription 1 (STAT1) in poly I:C-induced RAW 264.7 ( P < 0.05)., Conclusion: This study shows the inhibitory effect of emodin on poly I: C-induced RAW 264.7 via calcium-STAT pathway.

Objective: Our study aimed to investigate the possible modifying effects of leptin and combined use of resveratrol on rat renal I/R injury and their relationship on signal pathways and apoptosis-related mechanisms., Background: Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure., Methods: Male Sprague Dawley rats were divided into 5 groups: Control, I/R, I/R+leptin, I/R+resveratrol and I/R+leptin+resveratrol. Leptin (10 μg/kg BW) was administered (i.p.) 30 min prior to I/R. Resveratrol was administered by gavage at 20 mg/kg BW per d for 12 d prior to I/R. The left renal artery was exposed to 1 h of ischemia and 1 h of reperfusion., Results: Resveratrol treatment alone increased TNF-α, TNF-α R1, NF-κB, SIRT-1, STAT1 and STAT3 mRNA levels and decreased caspase 3 protein levels. Leptin treatment alone significantly decreased the caspase 3 protein levels. The combined use of resveratrol and leptin significantly increased STAT3, and caspase 3 mRNA levels, and decreased the caspase 3 protein levels. Apoptosis was significantly decreased especially in the leptin and leptin+resveratrol groups., Conclusion: The present study suggest that a combined use of resveratrol and leptin has preventive and regulatory effects on renal I/R injury; the mechanism involves decreasing apoptosis, likely by altering the JAK/STAT pathway and SIRT1 expression (Fig. 8, Ref. 24).

The present study was designed to assess the antiarthritic potential of ECF in collagen-induced arthritis (CIA) and explore its underlying mechanism. Methods. In vitro, lymphocyte proliferation assay was measured by CCK-8 kit. In vivo, the therapeutic potential of ECF on CIA was investigated; surface marker, Treg cell, and intracellular cytokines (IL-17A and IFN- γ ) were detected by flow cytometry. Th1 cell differentiation assay was performed, and mRNA expression in interferon- γ -related signaling was examined by q-PCR analysis. Results. In vitro, ECF markedly inhibited the proliferation of splenocytes in response to ConA and anti-CD3. In vivo, ECF treatment reduced the severity of CIA, inhibited IFN- γ and IL-6 secretion, and decreased the proportion of CD11b+Gr-1+ splenic neutrophil. Meanwhile, ECF treatment significantly inhibited the IFN- γ expression in CD4+T cell without obviously influencing the development of Th17 cells and T regulatory cells. In vitro, ECF suppressed the differentiation of naive CD4+ T cells into Th1. Furthermore, ECF intensely blocked the transcriptional expression in interferon- γ -related signaling, including IFN- γ , T-bet, STAT1, and STAT4. Conclusion. Our results indicated that ECF exerted antiarthritic potential in collagen-induced arthritis by suppressing Th1 immune response and interferon- γ -related signaling.

Background: Bee venom (BV), a type of toxin extracted from honeybees (Apis mellifera), has been empirically and widely used to treat inflammatory diseases throughout Asia. Essential BV (eBV) was developed by removing phospholipase A2 (PLA2) and histamine to lower occurrence of allergic reaction. This study investigated the anti-allergic and anti-inflammatory activities of eBV in vitro and in vivo and its underlying mechanism of action., Methods: The anti-inflammatory potential of eBV was assessed in vivo using a carrageenan-induced paw edema model. To further investigate the mechanism by which eBV exerts anti-allergic and anti-inflammatory effects, compound 48/80-stimulated RBL-2H3 cells and lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells were studied in vitro., Results: Release of β-hexosaminidase and histamine was increased by eBV in a dose-dependent manner, but these levels were lower in eBV compared to original BV at the same concentration. In addition, eBV suppressed compound 48/80-induced expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RBL-2H3 cells. eBV was also shown to suppress nitric oxide (NO) production by down-regulating mRNA expression and subsequent protein expression of inflammatory mediators in LPS-induced RAW 264.7 cells. Phosphorylation of activators and signal transducers of transcription 1/interferon regulatory factor 3 (STAT1/IRF3) was attenuated by eBV treatment. eBV significantly inhibited carrageenan-induced acute edema in vivo. Serum levels of prostaglandin E2 (PGE2), TNF-α, and IL-1β were also down-regulated by eBV., Conclusions: These results demonstrate that eBV inhibits allergic and inflammatory response by reducing inflammatory mediator production via regulation of the STAT1/IRF3 signaling pathway, suggesting that eBV is a feasible candidate for regulation of allergic-inflammatory response in complementary and alternative medicine., Competing Interests: The authors have declared that no competing interests exist.

CpG-oligodeoxynucleotide (CpG-ODN) is not only reported to protect against airway hyper responsiveness but is also known as a potent vaccine adjuvant for anti-tumor therapy. Little is known about the effect of CpG-ODN in mice with radiation-induced lung fibrosis (RILF), a common late stage form of tissue damage that occurs after thorax radiotherapy (RT). Here, we evaluated the immunomodulatory effects of CpG-ODN on the development of RILF. Mice were divided into four groups: (1) RT, single dose of 12Gy to the whole thorax; (2) CpG, only intraperitoneal injection of CpG-ODN for total 5 weeks; (3) RT+CpG, irradiation plus CpG-ODN treatment before and after irradiation for total 5 weeks; and (4) control (CTL): No RT or CpG-ODN treatment. In this study, we found that CpG-ODN treatment attenuated lung fibrosis and collagen deposition by increasing the number of M1 macrophagocytes, levels of Type-2 cytokines and TGF-β. CpG-ODN administration up-regulated the expression of TLR9 and STAT1 phosphorylation and reversed the expression of Type-2 immune response key transcription factor GATA-3. Activation of the JAK-STAT1 signaling pathway further enhanced M1 macrophage differentiation and Type-1 cytokine production. This study reveals the mitigating effect of early exposure to CpG-ODN on lung injury caused by irradiation in mice. The potential mechanism of action may be related to enhancement of Type-1 immunity. In conclusion, CpG-ODN may be a potential therapeutic target to treat RILF., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: Antinuclear antibodies (ANAs) are detected in ∼18% of females, yet autoimmune disease develops in only 5-8%. Immunologic differences between ANA-positive healthy individuals and patients with systemic lupus erythematosus (SLE) may elucidate the regulatory mechanisms by which ANA-positive individuals avoid transition to clinical autoimmune disease., Methods: Healthy individuals (n = 790) were screened for autoantibodies specific for 11 antigens associated with lupus, systemic sclerosis, and Sjögren's syndrome. From this screening, 31 European American ANA-positive healthy individuals were selected and demographically matched to ANA-negative controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency, and reactivity were analyzed by multiplex assays, immunophenotyping, and phosphospecific flow cytometry., Results: Of 790 individuals screened, 57 (7%) were ANA-positive. The majority of proinflammatory cytokines, including interferon-γ (IFNγ), tumor necrosis factor, interleukin-17 (IL-17), and granulocyte colony-stimulating factor, exhibited a stepwise increase in serum levels from ANA-negative controls to ANA-positive healthy individuals to SLE patients (P < 0.0001). IFNα, IFNβ, IL-12p40, and stem cell factor/c-Kit ligand were increased in SLE patients only (P < 0.05). B lymphocyte stimulator (BlyS) was elevated in SLE patients but decreased in ANA-positive individuals (P < 0.001). Further, IL-1 receptor antagonist (IL-1Ra) was down-regulated in SLE patients only (P < 0.0001). ANA-positive individuals had increased frequencies of monocytes, memory B cells, and plasmablasts and increased levels of pSTAT-1 and pSTAT-3 following IFNα stimulation compared with ANA-negative controls (P < 0.05)., Conclusion: ANA-positive healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated IFNs, BLyS, IL-12p40, and stem cell factor/c-Kit ligand. Further, severely decreased levels of IL-1Ra in SLE patients compared with ANA-positive individuals may contribute to disease development. These results highlight the importance of IFN-related pathways and regulatory elements in SLE pathogenesis., Competing Interests: No conflicts of interest to report, (© 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1.

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser(727) (but not Tyr(701)) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways.R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy., Significance Statement: Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity., (Copyright © 2016 the authors 0270-6474/16/363962-16$15.00/0.)

Background: Oestrogen receptor-negative (ER-) breast cancer is intrinsically sensitive to chemotherapy. However, tumour response is often incomplete, and relapse occurs with high frequency. The aim of this work was to analyse the molecular characteristics of residual tumours and early response to chemotherapy in patient-derived xenografts (PDXs) of breast cancer., Methods: Gene and protein expression profiles were analysed in a panel of ER- breast cancer PDXs before and after chemotherapy treatment. Tumour and stromal interferon-gamma expression was measured in xenografts lysates by human and mouse cytokine arrays, respectively., Results: The analysis of residual tumour cells in chemo-responder PDX revealed a strong overexpression of IFN-inducible genes, induced early after AC treatment and associated with increased STAT1 phosphorylation, DNA-damage and apoptosis. No increase in IFN-inducible gene expression was observed in chemo-resistant PDXs upon chemotherapy. Overexpression of IFN-related genes was associated with human IFN-γ secretion by tumour cells., Conclusions: Treatment-induced activation of the IFN/STAT1 pathway in tumour cells is associated with chemotherapy response in ER- breast cancer. Further validations in prospective clinical trials will aim to evaluate the usefulness of this signature to assist therapeutic strategies in the clinical setting.

The severity of intestinal inflammation in mammals can be profoundly impacted by weaning stress. Cathelicidins protect intestinal homeostasis by not only directly killing bacteria but also immune regulators. Here, we investigated the effects of cathelicidin-BF (C-BF) derived from the snake venoms of Bungarus fasciatus on weaning stress and intestinal inflammation and examined the mechanisms by which C-BF modulates intestinal immune responses in weanling piglets. We found that C-BF treatment significantly increased performance and reduced the diarrheal index in weanling piglets. Serum IL-6, IL-22 and TNF-α production was decreased by C-BF treatment. We demonstrated that C-BF inhibited the expression of the inflammatory cytokines TNF-α, IL-6 and IL-8 but increased the expression of the anti-inflammatory cytokine IL-10 in the intestine. We also demonstrated that C-BF suppressed inflammation by down-regulating the nuclear factor-κB (NF-κB) signaling pathway in the intestine and in LPS-induced macrophages in vitro. However, C-BF significantly induced the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) to enhance the phagocytosis of macrophages when inflammation was suppressed. In summary, our study demonstrated that C-BF suppressed intestinal inflammation by down-regulating the NF-κB signaling pathway and enhancing the phagocytosis of immune cells by activating STAT-1 during weaning., (Copyright © 2015. Published by Elsevier B.V.)

Tripchlorolide (T4), an extract of the natural herb Tripterygium wilfordii Hook F, has been found to possess anti-inflammatory and immunosuppressive actions. In the current study, these actions were evaluated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis by scoring the clinical signs, observing the infiltration of inflammatory cells and myelin sheath in the lumbar spinal cord of EAE mice. The results demonstrated that T4 (at a dose of 40 μg/kg) significantly reduced the severity of EAE and slowed down the ongoing EAE. Further analysis showed that T4 suppressed the mRNA and protein levels of the transcription factors T-bet and RoRrt and mRNA levels of IFN-γ and IL-17 in the spinal cords. Furthermore, T4 down-regulated the ERK1/2-NF-κB and JAK/STAT signaling pathways. At 40 μg/kg, T4 did not induce side effects on hematological parameters. These findings suggest that T4 ameliorates EAE by immunosuppression, providing a new insight into T4 application in multiple sclerosis treatment., (© 2015 International Society for Neurochemistry.)

Objective: To explore the effects of tanshinone and JAK2/STAT1 signaling pathway related mechanism in CVB3-induced myocarditis in murine., Methods: A total of 110 inbred male Balb/c mice which were 4 to 6 weeks-old were randomly divided into five groups: normal control (N, n = 10), myocarditis control (C, n = 25), tanshinone group (T, 15 mg · kg⁻¹ · d⁻¹, i.p., n = 25), janus kinase 2 inhibitor AG490 group (A, 10 mg · kg⁻¹ · d⁻¹, i.p., n = 25), T+A group (H, n = 25). Myocarditis was induced by 0.5 ml 10(-9.51) TCID50/ml CVB3 i.p. injection for 10 days in group C, T and H. Myocardial histopathologic changes were observed and phospho-STAT1 expressions were detected by immunohistochemistry and Western blot analysis. The levels of serum cardiac troponin I were detected with chemiluminescence immunoassay., Results: (1) Compared with group C, the histopathologic scores were significantly higher in group A and H (3.35 ± 0.57 and 3.34 ± 0.54 vs. 2.12 ± 0.39, P < 0.01), but lower in group T (1.40 ± 0.34 vs.2.12 ± 0.39, P < 0.01). (2) The expression of p-STAT1 protein was similar in group A and H compared to group N (P > 0.05), but was significantly lower than that in group C (0.017 ± 0.010 and 0.020 ± 0.010 vs. 0.246 ± 0.010, P < 0.01). The expression of p-STAT1 protein was significantly higher in group T than in group C (P < 0.01). (3) The levels of serum cardiac troponin I in group C, A, T and H were significantly higher than in group N ((0.42 ± 0.06), (1.17 ± 0.25), (0.23 ± 0.05) and (1.04 ± 0.19) µg/L vs. (0.02 ± 0.01) µg/L, all P < 0.01). The levels of serum cardiac troponin I were significantly higher in group A and H compared with group C ((1.17 ± 0.25) and (1.04 ± 0.19) µg/L vs. (0.42 ± 0.06) µg/L, P < 0.01), but were significantly lower in group T than in group C ((0.23 ± 0.05) µg/L vs. (0.42 ± 0.06) µg/L, P < 0.01). (4) There was a negative correlation between the expression level of p-STAT1 and the histopathologic scores (y = -4.503 x + 3.371, R² = 0.738, P < 0.01), but a positive correlation between the levels of serum cardiac troponin I and the histopathologic scores (y = 1.935x + 1.165, R² = 0.766, P < 0.01)., Conclusion: Tanshinone could attenuate myocardial injury via upregulating the JAK2/STAT1 signaling pathway in this murine viral myocarditis model.

Background: Inflammatory chemokines, such as macrophage-derived chemokine (MDC/CCL22), are elevated in the serum and lesioned skin of patients with atopic dermatitis (AD), and are ligands for C-C chemokine receptor 4, which is predominantly expressed on T helper 2 lymphocytes, basophils and natural killer cells. We have previously reported that quercetagetin has an inhibitory activity on inflammatory chemokines, which is induced by interferon (IFN)-γ and tumour necrosis factor (TNF)-α, occurring via inhibition of the signal transducer and activator of transcription 1 (STAT1) signal., Objectives: To investigate the specific mechanisms of quercetagetin on the STAT1 signal., Methods: We confirmed the inhibitory activity of quercetagetin on MDC and STAT1 in HaCaT keratinocytes. The interaction between STAT1 and IFN-γR1 was investigated using immunoprecipitation. The small interfering RNA approach was used to investigate the role of suppressor of cytokine signalling 1 (SOCS1) and transforming growth factor (TGF)-β1 induced by quercetagetin., Results: Quercetagetin inhibited the expression of MDC at both the protein and mRNA levels in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes. Moreover, quercetagetin inhibited the phosphorylation of STAT1 through upregulation of SOCS1. Increased expression of SOCS1 disrupted the binding of STAT1 to IFN-γR1. Furthermore, quercetagetin augmented the expression of TGF-β1, which is known to modulate the immune response and inflammation., Conclusions: These results suggest that quercetagetin may be a potent inhibitor of the STAT1 signal, which could be a new molecular target for anti-inflammatory treatment, and may thus have therapeutic applications as an immune modulator in inflammatory diseases such as AD., (© 2014 British Association of Dermatologists.)

Purpose: The consequences of precipitously rising allergic skin inflammation rates worldwide have accelerated the risk of atopic dermatitis (AD). Natural product-based agents with good efficacy and low risk of side effects offer promising prevention and treatment strategies for inflammation-related diseases. We have already reported that Platycodon grandiflorum root-derived saponins (Changkil saponins, CKS) have many pharmacological effects, including anti-inflammatory and anti-allergic effects, but its influence on AD remains unclear. Therefore, we evaluated the inhibitory effect of CKS, mainly platycodin D, on AD-like skin symptoms in mice and the possible mechanisms in cells., Methods: Mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNCB). Four weeks after challenge, mice were treated with oral administration of CKS for 4 weeks. In addition, cells were used to evaluate the effect of CKS, mainly platycodin D, on the TARC expression regulated mechanism., Results: CKS attenuated DNCB-induced dermatitis severity, serum levels of IgE and TARC, and mRNA expression of TARC, TNF-α, IFN-γ, IL-4, IL-5, and IL-13 in mice. Histopathological examination showed reduced thickness of the epidermis/dermis and dermal infiltration of inflammatory cells and mast cells in the ears. Moreover, CKS and platycodin D inhibited TNF-α/IFN-γ-induced TARC expression through the suppression of NF-κB and STAT1 and induction of Nrf2/ARE-mediated hemeoxygenase-1 (HO-1) expression in cells., Conclusion: We suggest that CKS and platycodin D inhibited the development of AD-like skin symptoms by regulating cytokine mediators and may be an effective alternative therapy for AD-like skin symptoms., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Garcinol (1), a polyisoprenylated benzophenone occurring in Garcinia species, has been reported to exert anti-inflammatory activity in LPS-stimulated macrophages, through inhibition of NF-κB and/or JAK/STAT-1 activation. In order to provide deeper insight into its effects on the cytokine signaling pathway and to clarify the underlying molecular mechanisms, 1 was isolated from the fruits of Garcinia cambogia along with two other polyisoprenylated benzophenones, guttiferones K (2) and guttiferone M (3), differing from each other in their isoprenyl moieties and their positions on the benzophenone core. The affinities of 1-3 for the STAT-1 protein have been evaluated by surface plasmon resonance and molecular docking studies and resulted in KD values in the micromolar range. Consistent with the observed high affinity toward the STAT-1 protein, garcinol and guttiferones K and M were able to modulate cytokine signaling in different cultured cell lines, mainly by inhibiting STAT-1 nuclear transfer and DNA binding, as assessed by an electrophorectic mobility shift assay.

Background and Objective: Host modulatory agents directed at inhibiting specific proinflammatory mediators could be beneficial in terms of attenuating periodontal disease progression and potentially enhancing therapeutic responses. The aim of this study was to investigate whether daidzein could modulate the production inflammatory mediators in macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and to delineate underlying mechanisms of action., Material and Methods: LPS was extracted from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The amounts of nitric oxide (NO) and interleukin-6 (IL-6) secreted into the culture medium were assayed. A real-time PCR was performed to quantify inducible nitric oxide synthase (iNOS) and IL-6 mRNA expression. We used immunoblot analysis to characterize iNOS protein expression, phosphrylation of c-Jun N-terminal kinase (JNK) and p38, degradation of inhibitory κB-α (IκB-α), nuclear translocation of nuclear factor-κB (NF-κB) subunits and phosphorylation of signal transducer and activator of transcription 1 (STAT1). The DNA-binding activity of NF-κB was assessed by using ELISA-based kits., Results: Daidzein significantly inhibited the production of NO and IL-6, as well as their mRNA expression, in P. intermedia LPS-treated RAW264.7 cells. The JNK and p38 pathways were not involved in the regulation of LPS-induced NO and IL-6 release by daidzein. Daidzein inhibited the degradation of IκB-α induced by P. intermedia LPS. In addition, daidzein suppressed NF-κB transcriptional activity via regulation of the nuclear translocation and DNA-binding activity of NF-κB p50 subunit and blocked STAT1 phosphorylation., Conclusion: Although additional studies are required to dissect the molecular mechanism of action, our results suggest that daidzein could be a promising agent for treating inflammatory periodontal disease. Further research in animal models of periodontitis is necessary to better evaluate the potential of daidzein as a novel therapeutic agent to treat periodontal disease., (© 2011 John Wiley & Sons A/S.)

Background: The mechanisms responsible for the accelerated cardiovascular disease in diabetes, as well as the increased hypertrophic effects of angiotensin II (Ang II) under hyperglycemic condition, are not very clear. Evidences show that platelet-derived growth factor (PDGF) and protein kinase C (PKC) play a critical role in this effect. In our study, we examined the role of PKC and PDGF receptor on JAK2 and STAT1 phosphorylation under high glucose (HG) condition (25 mmol/L) in response to Ang II in cultured vascular smooth muscle cells (VSMC)., Methods: VSMCs were isolated from the thoracic aorta of male Wistar rats and were cultured. Growth-arrested VSMCs were placed in either normal glucose (NG) or HG condition for 48 h and then VSMCs were stimulated with agonists and antagonists. The tyrosine phosphorylation of JAK2 or STAT were determined by immunoblotting using specific antibodies., Results: High glucose markedly increased the phosphorylation of tyrosine residues of JAK2 and serine residues of STAT 1 compared with cells cultured in NG (5.5 mmol/L) with and without Ang II stimulation. Experiments made with specific PDGF-β receptor inhibitor AG1295 and PKC inhibitor GF109203X showed that there were no changes in Ang II-stimulated JAK2 and STAT1 phosphorylation under NG and HG conditions compared with experiments without inhibitors., Conclusion: According to our findings, Ang II-stimulated JAK2 and STAT1 phosphorylation under either NG or HG condition do not proceed via a different pathway rather than PKC and PDGF-β receptor.

Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative disease by producing excessive proinflammatory cytokines and nitric oxide (NO). Luteolin, a naturally occurring polyphenolic flavonoid antioxidant, has potent anti-inflammatory and neuroprotective properties both in vitro and in vivo. However, the molecular mechanism of luteolin-mediated immune modulation in microglia is not fully understood. In the present study, we report the inhibitory effect of luteolin on lipopolysaccharide (LPS)/interferon γ (IFN-γ)-induced NO and proinflammatory cytokine production in rat primary microglia and BV-2 microglial cells. Luteolin concentration-dependently abolished LPS/IFN-γ-induced NO, tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) production as well as inducible nitric oxide synthase (iNOS) protein and mRNA expression. Luteolin exerted an inhibitory effect on transcription factor activity including nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF-1) in LPS/IFN-γ-activated BV-2 microglial cells. Biochemical and pharmacological studies revealed that the anti-inflammatory effect of luteolin was accompanied by down-regulation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), Akt and Src. Further studies have demonstrated that the inhibitory effect of luteolin on intracellular signaling execution and proinflammatory cytokine expression is associated with resolution of oxidative stress and promotion of protein phosphatase activity. Together, these results suggest that luteolin suppresses NF-κB, STAT1 and IRF-1 signaling, thus attenuating inflammatory response of brain microglial cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several malignant diseases, including Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman's disease. The objectives of this study were to investigate the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) against KSHV early lytic genes and to assess their efficacy in severe combined immunodeficiency disease (SCID) mice against PEL engraftment. PPMOs are short, single-stranded DNA analogues that contain a backbone of morpholine rings and phosphorodiamidate linkages and have high delivery efficiency into cells., Methods: PEL cells were treated with PPMOs against viral interferon regulatory factor 1 (vIRF-1) and expression of vIRF-1 was analysed. PPMOs against vIRF-1 and viral interleukin-6 (vIL-6) were evaluated against PEL cell engraftment in SCID mice. The PPMOs were incubated with BCBL-1 cells and then introduced into the peritoneal cavities of SCID mice, followed by 9 more doses of PPMOs administered at 2-day intervals. At weeks 3 and 9 after BCBL-1 delivery, peritoneal lavage was collected and the ratio of PEL cells among total cells was determined by flow cytometry analysis., Results: Treatment of PEL cells with PPMOs against vIRF-1 led to a reduction of vIRF-1 expression in a dose-dependent manner. Reduction of vIRF-1 expression resulted in higher levels of cellular interferon regulatory factor 3 and of signal transducer and activator of transcription 1. SCID mice receiving a PPMO against vIL-6 had no engraftment of PEL cells and remained healthy throughout the 120-day study., Conclusions: This study showed that PPMOs can be effective antiviral agents against KSHV. Blocking the expression of early lytic genes might be beneficial for the control of KSHV-associated malignant diseases.

Aims: C-peptide is a product of pro-insulin cleavage. Numerous studies have demonstrated that C-peptide, although not influencing blood glucose control, may play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. The aim of this paper was to present a novel function of C-peptide, focusing on its role in nitric oxide (NO) generation., Main Methods: Murine macrophage Raw264.7 cells and primary peritoneal macrophages were incubated under control conditions, or with C-peptide. Expression level of iNOS and phosphorylation status of JAK2/STAT1 were analyzed by Western blot. Fluorometric NO assay kit was used to assess the concentration of nitrite in culture medium. Intracellular calcium concentration was measured with calcium indicator dyes, such as Fura-2 and Fluo-3 AM., Key Findings: C-peptide increased the level of nitrites in murine macrophage Raw264.7 cells. The nitrites production induced by lipopolysaccharide (LPS) was further enhanced by co-treatment of C-peptide. This up-regulation of nitrites generation also correlated with the induction of inducible nitric oxide synthase (iNOS), a prominent marker of macrophage activation. In addition, C-peptide increased the intracellular concentration of calcium levels. Moreover, C-peptide-induced nitrites generation and increase in calcium was observed in freshly isolated primary peritoneal macrophages. In addition, C-peptide specifically affected the Janus activated kinase (JAK)/signal transducer and activated transcription (STAT) pathway. Finally, C-peptide-mediated nitrites generation and JAK2/STAT1 phosphorylation were not detected in the presence of the intracellular calcium chelator, BAPTA-AM., Significance: These results suggest that C-peptide may elicit immune modulatory function via modulation of the calcium/JAK-STAT pathway.

The transcription factor STAT1 is involved in signal transduction of type I and II interferons (IFNs). However, the molecular characteristics of the STAT1 regulatory region still remain to be elucidated in teleosts. In the present study, the complete cDNA and the regulatory region of the STAT1 gene were isolated from snakehead (Channa argus). More than 2.4kb 5'-flanking region of STAT1 shares the regulatory elements of IFN-stimulated response element (ISRE) and IFN-gamma activation site (GAS). Consensus ISRE and GAS were located from -373 to -361 and -716 to -724 in the promoter region, respectively. Moreover, it is noticeable that the crucial elements of ISRE (+698 to +710) and GAS (+294 and +301) are present in the first intron of snakehead STAT1. Comparisons of six vertebrate STAT1 5'-flanking regions all present the common sequence characteristics of IFN-induced gene promoter, which include ISRE, GAS and Sp1 sites. In order to further characterize the snakehead STAT1 regulatory region, six reporter constructs of snakehead STAT1 promoter and first intron were generated to examine the specificity to human interferon-gamma (hIFN-gamma). Only those constructs containing the ISRE element showed notable reporter activity after stimulation of Hela cells with hIFN-gamma. However, sequential deletions of putative transcription factor binding sites indicated that GAS elements have little effect on the promoter and intronic activity in response to hIFN-gamma. Taken together, these results suggest that the regulatory mechanisms of IFN-signalling appear to be mediated in a similar manner in fish and mammals., (Copyright 2009 Elsevier B.V. All rights reserved.)

IFN-beta is an approved therapeutic option for the treatment of multiple sclerosis. The molecular mechanisms underlying the effects of IFN-beta in multiple sclerosis are not fully understood. Migration of dendritic cells (DCs) from the inflammatory site to draining lymph nodes for Ag presentation and activation of naive T cells and to the CNS for reactivation of encephalitogenic T cells requires CCR7 and matrix metalloproteinase (MMP)-9 expression. This article reports for the first time that IFN-beta inhibits CCR7 expression and MMP-9 production in mature DCs and reduces their migratory capacity. The effect of IFN-beta is mediated through STAT-1. In vivo treatment with IFN-beta results in lower numbers of DCs migrating to the draining lymph node following exposure to FITC and in reduced expression of CCR7 and MMP-9 in splenic CD11c(+) DCs following LPS administration. IFN-beta and IFN-gamma share the same properties in terms of their effects on CCR7, MMP-9, and DC migration, but they have opposite effects on IL-12 production. In addition, IFN-beta-treated DCs have a significantly reduced capacity for activating CD4(+) T cells and generating IFN-gamma-producing Th1 cells. The suppression of mature DC migration through negative regulation of CCR7 and MMP-9 expression represents a novel mechanism for the therapeutic effect of IFN-beta.

Background and Purpose: Benzoxathiolone derivatives have shown anti-inflammatory and immunomodulatory potential in acne and psoriatic disorders. However, little is known about the molecular basis for these pharmacological effects. In this study, we decided to investigate the anti-inflammatory actions of a benzoxathiolone derivative LYR-71, 6-methyl-2-propylimino-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one, in interferon (IFN)-gamma-activated macrophages., Experimental Approach: RAW 264.7 macrophages or primary macrophages, derived from bone marrow of C3H/HeJ mice, were stimulated with IFN-gamma in the presence of LYR-71. Nitric oxide (NO) or chemokine production was measured by Griess reaction or enzyme-linked immunosorbent assay. RAW 264.7 cells were used to examine the molecular mechanisms of LYR-71 in modulating IFN-gamma-induced inflammatory responses., Key Results: LYR-71 down-regulated IFN-gamma-induced transcription of inducible NO synthase, IFN-gamma-inducible protein-10 and the monokine induced by IFN-gamma genes in macrophages. This effect was mediated by uncoupling tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)-1 in response to IFN-gamma. LYR-71 directly inhibited the in vitro catalytic activity of Janus kinase (JAK)-2. Further, the inhibitory actions of LYR-71 on IFN-gamma-induced STAT-1 phosphorylation and NO production were consistently abolished in the presence of peroxyvanadate, implying another target dependent on protein tyrosine phosphatase., Conclusions and Implications: Taken together, LYR-71 could restrain IFN-gamma-induced inflammatory responses through uncoupling the tyrosine phosphorylation of STAT-1, an activation index of JAK-STAT-1 signalling, in macrophages. These results may provide a molecular mechanism underlying anti-inflammatory actions shown by benzoxathiolone derivatives.

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-kappaB signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-kappaB p65 and modifies nuclear NF-kappaBp50. Here, we demonstrated that both nuclear STAT-1 and NF-kappaB are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-kappaB activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-kappaB proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-kappaB p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN-gamma production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-kappaB. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-kappaB small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-kappaB in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-kappaB proteins.

IFN-beta-1a has been used over the past 15 years as a primary therapy for relapsing-remitting multiple sclerosis (MS). However, the immunomodulatory mechanisms that provide a therapeutic effect against this CNS inflammatory disease are not yet completely elucidated. The effect of IFN-beta-1a on Th17 cells, which play a critical role in the development of the autoimmune response, has not been extensively studied in humans. We have investigated the effect of IFN-beta-1a on dendritic cells (DCs) and naive CD4(+)CD45RA(+) T cells derived from untreated MS patients and healthy controls in the context of Th17 cell differentiation. We report that IFN-beta-1a treatment down-regulated the expression of IL-1beta and IL-23p19 in DCs, whereas it induced the gene expression of IL-12p35 and IL-27p28. We propose that IFN-beta-1a-mediated up-regulation of the suppressor of cytokine signaling 3 expression, induced via STAT3 phosphorylation, mediates IL-1beta and IL-23 down-regulation, while IFN-beta-1a-induced STAT1 phosphorylation induces IL-27p28 expression. CD4(+)CD45RA(+) naive T cells cocultured with supernatants from IFN-beta-1a-treated DCs exhibited decreased gene expression of the Th17 cell markers retinoic acid-related orphan nuclear hormone receptor c (RORc), IL-17A, and IL-23R. A direct IFN-beta-1a treatment of CD45RA(+) T cells cultured in Th17-polarizing conditions also down-regulated RORc, IL-17A, and IL-23R, but up-regulated IL-10 gene expression. Studies of the mechanisms involved in the Th17 cell differentiation suggest that IFN-beta-1a inhibits IL-17 and induces IL-10 secretion via activated STAT1 and STAT3, respectively. IFN-beta's suppression of Th17 cell differentiation may represent its most relevant mechanism of selective suppression of the autoimmune response in MS.

Background: Diabetes increases the risk for fatal myocardial infarction and development of heart failure. Levosimendan, an inodilator acting both via calcium sensitization and opening of ATP-dependent potassium channels, is used intravenously for acute decompensated heart failure. The long-term effects of oral levosimendan on postinfarct heart failure are largely unknown., Objective: To examine whether oral treatment with levosimendan could improve cardiac functions and prevent cardiac remodeling after myocardial infarction in a rodent model of type 2 diabetes, the Goto-Kakizaki rat., Methods: Myocardial infarction (MI) was induced to diabetic Goto-Kakizaki and nondiabetic Wistar rats by coronary ligation. Twenty-four hours after surgery, Goto-Kakizaki and Wistar rats were randomized into four groups: MI group without treatment, MI group with levosimendan for 12 weeks (1 mg/kg per day), sham-operated group, sham-operated group with levosimendan. Blood pressure, cardiac functions as wells as markers of cardiac remodeling were determined., Results: In Goto-Kakizaki rats, MI induced systolic heart failure, pronounced cardiac hypertrophy in the remote area, and sustained cardiomyocyte apoptosis. Postinfarct cardiac remodeling was associated with increased atrial natriuretic peptide, interleukin-6 and connective tissue growth factor mRNA expressions, as well as three-fold increased cardiomyocyte senescence, measured as cardiac p16 mRNA expression. Levosimendan improved cardiac function and prevented postinfarct cardiomyocyte hypertrophy, cardiomyocyte apoptosis, and cellular senescence. Levosimendan also ameliorated MI-induced atrial natriuretic peptide, IL-6, and connective tissue growth factor overexpression as well as MI-induced disturbances in calcium-handling proteins (SERCA2, Na-Ca exchanger) without changes in diabetic status or systemic blood pressure. In nondiabetic Wistar rats, MI induced systolic heart failure; however, the postinfarct cardiac remodeling was associated with less pronounced cardiac hypertrophy, cardiomyocyte apoptosis, inflammatory reaction, and induction of cellular senescence. Levosimendan only partially prevented postinfarct heart failure and cardiac remodeling in Wistar rats., Conclusion: Our findings suggest a therapeutic role for oral levosimendan in prevention of postinfarct heart failure and cardiac remodeling in type 2 diabetes and underscore the importance of sustained cardiomyocyte apoptosis and induction of cellular senescence in the pathogenesis.

Members of the protein kinase C (PKC) family are activated by interferon-gamma (IFN-gamma) and modulate IFN-gamma-induced cellular responses by regulating the activity of transcription factors. We previously reported that PKC-alpha enhances the ability of IFN regulatory factor-1 to transactivate the class II transactivator (CIITA) promoter IV in IFN-gamma-stimulated macrophages. In addition, we showed that IFN-gamma induces the nuclear translocation of PKC-alpha but the mechanisms for this remain to be elucidated. In this study, we sought to identify signalling pathways involved in IFN-gamma-induced activation of PKC-alpha and to characterize their potential roles in modulating IFN-gamma-induced responses in macrophages. IFN-gamma-mediated nuclear translocation of PKC-alpha was a Janus activated kinase 2 (JAK2)-independent process, which required phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). However, PKC-alpha phosphorylation was independent of PI3K and p38 MAPK, indicating that IFN-gamma-induced phosphorylation and nuclear translocation of PKC-alpha are mediated by distinct mechanisms. In addition, inhibition of PI3K, but not of p38 MAPK, strongly impaired IFN-gamma-induced CIITA and MHC II gene expression. Finally, PKC-alpha associated with signal transducer and activator of transcription 1 (STAT1) and was required for the phosphorylation of STAT1 on serine 727 in IFN-gamma-stimulated macrophages. Taken together, our data indicate that PI3K and p38 MAPK modulate IFN-gamma-stimulated PKC-alpha nuclear translocation independently of JAK2 activity and that both PI3K and PKC-alpha are required for type IV CIITA and MHC II gene expression in IFN-gamma-stimulated macrophages.

Ex vivo analysis of signaling pathways operating in tumor tissue is complicated by the three-dimensional structure, in particular by stroma-epithelial interactions. Studies performed with pure populations of tumor cells usually do not take into account this issue. One possibility to preserve the tissue architecture is the use of tumor slices. However, diffusion of oxygen and nutrients may become limiting factors, resulting in decreased cell viability and change of tissue morphology, especially after long-term incubation of slices. By using precision cut slices of defined thickness, we were able to establish culture conditions for tumor material obtained from MMTV-neu transgenic mice, which allow the study of the action of cytokines and cytotoxic drugs for up to 24 h. A slice thickness of 160 mum was found to be optimal for viability and handling of material. These slices were highly responsive to the action of the cytokine IFN-gamma, as evident form the increase of pY701 STAT1, detected by both immunohistochemistry and western blotting, and by the increase of mRNA levels of the IFN-gamma response genes IRF-1, SOCS-1, and STAT1, analyzed by reverse transcriptase-polymerase chain reaction. Furthermore, induction of apoptosis and increase of DNA damage could be monitored after treatment with IFN-gamma or doxorubicin. The slices were also a convenient source for the establishment of explant cultures of tumor epithelial cells. It is concluded that cultivation of precision-cut tumor slices provides a convenient way for the ex vivo molecular analysis of MMTV-neu tumor tissue under conditions which closely simulate the situation in vivo and can provide an alternative to in vivo experiments.

To investigate abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR) expression in chronic inflammatory airway diseases and its regulation mechanisms, the present study was designed to observe the expression of CFTR, CFTR chloride current and the possible relevant signal pathways in in vitro and in vivo bronchial epithelium by using real-time PCR, immunofluorescence, Western blot and whole cell patch-clamp. The results demonstrated that CFTR staining was decreased in rat airway epithelium under ozone stress. Ozone stress also down-regulated CFTR protein and mRNA expression and CFTR chloride current in cultured human bronchial epithelial cells (HBEC). STAT1 signal pathway was checked to investigate the signal mechanism. It was found that pretreatment with STAT1 inhibitor attenuated the down-regulated CFTR expression induced by ozone stress. We also observed that ozone stress accelerated the phosphorylation of STAT1 in HBEC, which could be influenced by some signaling molecules related to the early transduction of cellular stress. Furthermore, reactive oxygen species inhibitors N-acetylcysteine and nitric oxide synthase inhibitor aminoguanidine increased the expression of CFTR. Ozone stress could down-regulate the expression of CFTR and decrease CFTR chloride current in HBEC. The signal mechanism which referred to cascade events in cells included early oxidative stress signal transmission molecules, and subsequently transcription modulator STAT1.

Intravenous (i.v.) administration of encephalitogenic peptide can effectively prevent experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis; however, the underlying cellular and molecular mechanisms are not fully understood. In this study, we induced i.v. tolerance to EAE by administration of MOG(35-55) peptide and determined the effect of this approach on intracellular signaling pathways of the IL-23/IL-17 system, which is essential for the pathogenesis of MS/EAE. In tolerized mice, phosphorylation of JAK/STAT-1, -4, ERK1/2 and NF-kappaBp65 were significantly reduced in splenocytes and the central nervous system. MOG i.v. treatment led to significantly lower production of IL-17, and administration of exogenous IL-17 slightly broke immune tolerance, which was associated with reduced activation of STAT4 and NF-kappaB. Suppressed phosphorylation of these pathway molecules was primarily evident in CD11b(+) and small numbers of CD4(+), CD8(+) and CD11c(+) cells. More importantly, adoptive transfer of CD11b(+) splenocytes of tolerized mice effectively delayed onset and reduced clinical severity of actively induced EAE. This study correlates MOG i.v. tolerance with modulation of Jak/STAT signaling pathways and investigates novel therapeutic avenues for the treatment of EAE/MS.

The steroid hormone 20-hydroxyecdysone (20HE) is an essential signaling molecule that modulates molting response in insects and may function as a putative anabolic factor in vertebrate animals, although no mammalian 20HE receptor has been identified. Here we show that in H4IIE cell culture, 20HE treatment decreased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), reduced glucose production, and induced Akt2 phosphorylation sensitive to the phosphoinositide-3 kinase pathway-specific inhibitor LY-294002. Daily oral administration of 20HE (10 mg/kg for 13 wk) ameliorated obesity and insulin resistance in C57BL/6J mice fed a high-fat diet and produced a significant decrease of body weight gain and body fat mass compared with nontreated animals as demonstrated by dual-energy X-ray absorptiometry analysis. In addition, plasma insulin levels and glucose tolerance were significantly lowered by 20HE treatment. These changes were accompanied by the reduced hepatic expression of PEPCK and G6Pase and increased adiponectin production by visceral fat tissue. These studies demonstrate the anti-obesity and anti-diabetic effects of 20HE and begin to elucidate its putative cellular targets both in vitro and in vivo.

Background: Apoptosis is involved in acute renal failure (ARF). Its exact mechanism still remains to be explored. The Jak-Stat pathway participates in inflammation, apoptosis and tumorigenesis. In an in vitro model of renal ischemia/reperfusion injury (IRI), we investigated the role of Jak2 kinase inhibition on signal transducer and activator of transcription 1 (Stat1) and Stat3 activations as well as apoptosis of human proximal tubular epithelial cells (HKCs) induced by adenosine triphosphate (ATP) depletion/recovery., Methods: ATP depletion of HKCs is induced by antimycin A., Results: The Jak2-specific inhibitor AG490 decreased Stat1 and Stat3 phosphorylations and promoted HKC apoptosis induced by ATP depletion/recovery., Conclusions: Our results have demonstrated that Jak2 inhibition participated in the ATP depletion-induced apoptosis of HKCs, which might be a potential target for prevention and treatment of ARF.

Ethyl pyruvate (EP) has been demonstrated to have an anti-inflammatory function. However, the molecular mechanisms underlying the anti-inflammatory action of EP are largely unknown. We here show that EP exerts its anti-inflammatory effect by inhibiting ROS-dependent STAT signaling through its antioxidant activity, like vitamin C or N-acetyl-L-cysteine. The inhibition of STAT1 and STAT3 by EP prevented their translocation to the nucleus and consequently inhibited expression of iNOS and COX-2 by inhibiting STAT1- and STAT3-mediated transcriptional activity, followed by changes in chromatin conformation via deacetylation of histones H3 and H4 in both gene promoters. EP also suppressed transcripts of other STAT-responsive inflammatory genes such as IL-1beta, IL-6, TNF-alpha, and MCP-1. We further found that the mechanism of inhibition of STAT1 and STAT3 by EP is due to inhibition of JAK2 through Rac1 inactivation and SOCS1 induction. These findings offer new therapeutic possibilities for EP based on a better understanding of the mechanism underlying the action of EP.

Background: Acute alcohol consumption is associated with induction of immuno-inhibitory cytokines and down-regulation of pro-inflammatory responses to various pathogens. We previously reported that alcohol activates janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling leading to IL-10 induction. The JAK-STAT pathway also activates its own negative regulators, suppressors of cytokine signaling (SOCS) 1 and SOCS3. SOCS proteins are inducible inhibitors that negatively regulate STAT3/STAT1 signaling pathways induced by cytokines, IL-6 or IFNs. Here we aimed to explore the effect of acute alcohol on induction of SOCS1/SOCS3 and regulation of STAT3/STAT1 pathways induced by IL-6 or IFNs in human monocytes., Methods: Blood samples from normal volunteers were collected before and 24 hours after consumption of 2 ml vodka/kg body weight. For in vitro experiments human monocytes were pretreated with ethanol (EtOH) followed by stimulation with cytokines; proteins were analyzed by Western blot, nuclear protein binding to DNA by EMSA, and RNA by real time PCR., Results: Acute in vivo or in vitro alcohol treatment increased both SOCS1 and SOCS3 RNA expression in monocytes. Alcohol treatment resulted in increased STAT3 and STAT1 DNA binding capacity. Activation of both STAT1 and STAT3 has been shown to induce SOCS1/3. We hypothesized that induction of SOCS proteins by alcohol in turn may lead to modulation of cytokine signaling through STAT1 and STAT3. Indeed, we observed significant down-regulation of IL-6-, IFNalpha- and IFNgamma-induced STAT1 DNA binding as well as inhibition of IL-6- and IFNgamma-induced STAT3 when alcohol was added to monocytes 3 hours prior to the cytokine stimulation. Consistent with inhibition of IL-6-induced STAT3 DNA binding in alcohol-pretreated cells, the levels of IL-6-dependent genes, MCP-1 and ICAM-1, was reduced after IL-6 stimulation. Similar to EtOH alone, combined EtOH+IL-6 simulation resulted in increased expression of both SOCS3 and SOCS1 genes., Conclusion: While acute alcohol treatment alone activates STAT1/3 signaling pathways and induces SOCS3 and SOCS1 levels in monocytes, alcohol also leads to down-regulation of IL-6-, IFNalpha-, and IFNgamma-induced signaling via STAT1/STAT3 pathways, likely through excessive SOCS activation.

Interferon alpha (IFN-alpha) not only plays a key role in innate host immunity against infections but also is involved in the cellular functions of the central nervous system (CNS). In this study, we examined the impact of morphine on IFN-alpha expression in human neuronal cells (NT2-N). Similar to human immune cells, NT2-N cells also expressed IFN-alpha at both mRNA and protein levels. IFN-alpha expression in NT2-N cells, however, was inhibited by morphine. Naltrexone antagonized the inhibitory effect of morphine on IFN-alpha expression in NT2-N cells. The specific mu opioid receptor antagonist, Cys2, Tyr3, Arg5, Pen7-amide (CTAP), also blocked the morphine action on intracellular IFN-alpha expression. Investigation of the mechanisms involved in the morphine action showed that although morphine had little effect on the expression of key IFN regulatory factors (IRFs), morphine inhibited IFN-alpha promoter activation and suppressed the expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1) in the neuronal cells. These findings provide direct in vitro evidence that opioids may impair neuronal cell-mediated innate protection in the CNS.

In this study, we evaluated the activity of two novel pyrazolopyrimidine derivatives (Si 34 and Si 35) against ARO cells, a human anaplastic thyroid cancer cell line. ARO cells exposed to different concentrations of the drugs showed a reduced growth rate and an increase of mortality. After 72 h incubation, doses of 5 and 10 microM Si 34 determined a decrease of cell counts by approximately 25% and approximately 75% compared with those of control cells respectively. Similar findings were observed using Si 35. Treatment with both Si 34 and Si 35 at 10 microM increased cell mortality also ( approximately 29% and approximately 18% respectively). At these concentrations, a decrease in cyclin D1 levels was observed. To improve the biopharmaceutical properties, a liposome formulation was prepared. When entrapped in unilamellar liposomes, Si 34 exerted its cytotoxic effects even at lower doses (maximal inhibition at 5 microM) and after shorter incubation time (48 h) either in ARO or other thyroid cancer cell lines. The effects were associated with weak apoptotic death. Inhibition of epidermal growth factor-stimulated src and ERK phosphorylation, as well as reduction of migration properties of ARO cells was also observed. Moreover, the growth of tumor xenografts induced in severe combined immunodeficiency (SCID) mice was inhibited by i.v. administration of 25-50 mg/kg of the drug liposomal formulation. In conclusion, the liposomal preparation of this novel pyrazolopyrimidine derivative appears to be a promising tool for the treatment of anaplasic thyroid cancer.

Interferon-beta (IFN-beta) reduces disease activity in a subgroup of patients with relapsing remitting multiple sclerosis (MS). The mechanism of action as well as the pathophysiological basis of responsiveness to IFN-beta is not well understood. Since T-cell activation plays an important part in the pathophysiology of MS, we here investigated the effect of IFN-beta on the expression of co-signaling pathways (CD28-CD80/CD86, CD154-CD40, ICOS-ICOSL, PD-1-PD-L1/2) in MS patients and correlated the results with the clinical response to IFN-beta in individual patients. Expression of co-signaling molecules was measured by flow cytometry in vitro on peripheral blood mononuclear cells after incubation with IFN-beta, and in vivo in whole blood samples of 32 untreated and 24 IFN-beta treated MS patients, including 13 patients longitudinal. IFN-beta treatment induced upregulation of CD40, CD80, CD86, PD-L1 and PD-L2 on monocytes as well as PD-L1 on CD4+-T-cells in vitro and in vivo. IFN-beta treated MS patients were grouped into responders and non-responders on the basis of Kurtzkés EDSS (expanded disability status scale) progression and relapse rate. Upregulation of CD40, CD86 and PD-L2 on monocytes was associated with treatment response to IFN-beta (P < 0.001, P = 0.028 and P = 0.028, respectively). Our results show that IFN-beta upregulates co-stimulatory as well as co-inhibitory molecules in vitro and in vivo implicating that modulation of the balance between positive and negative co-stimulatory signals might be an important part of the mechanism of action of IFN-beta in MS. Upregulation of the expression of CD40, CD86 and PD-L2 may be useful as a predictive marker for clinical response to IFN-beta treatment at early timepoints during IFN-beta therapy.