Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

PubMed

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

PROBLEM OF FORMING IN A MAN-OPERATOR A HABIT OF TRACKING A MOVING TARGET,

DTIC Science & Technology

Cybernetics stimulated the large-scale use of the method of functional analogy which makes it possible to compare technical and human activity systems...interesting and highly efficient human activity because of the psychological control factor involved in its operation. The human tracking system is

Nuclear Receptor Activity and Liver Cancer Lesion Progression

EPA Science Inventory

Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. We explored this question using human CAR, PXR, PPARα,...

Dexamethasone inhibits IL-12p40 production in lipopolysaccharide-stimulated human monocytic cells by down-regulating the activity of c-Jun N-terminal kinase, the activation protein-1, and NF-kappa B transcription factors.

PubMed

Ma, Wei; Gee, Katrina; Lim, Wilfred; Chambers, Kelly; Angel, Jonathan B; Kozlowski, Maya; Kumar, Ashok

2004-01-01

IL-12 plays a critical role in the development of cell-mediated immune responses and in the pathogenesis of inflammatory and autoimmune disorders. Dexamethasone (DXM), an anti-inflammatory glucocorticoid, has been shown to inhibit IL-12p40 production in LPS-stimulated monocytic cells. In this study, we investigated the molecular mechanism by which DXM inhibits IL-12p40 production by studying the role of the mitogen-activated protein kinases (MAPKs), and the key transcription factors involved in human IL-12p40 production in LPS-stimulated monocytic cells. A role for c-Jun N-terminal kinase (JNK) MAPK in LPS-induced IL-12p40 regulation in a promonocytic THP-1/CD14 cell line was demonstrated by using specific inhibitors of JNK activation, SP600125 and a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase-1 mutant. To identify transcription factors regulating IL-12p40 gene transcription, extensive deletion analyses of the IL-12p40 promoter was performed. The results revealed the involvement of a sequence encompassing the AP-1-binding site, in addition to that of NF-kappaB. The role of AP-1 in IL-12p40 transcription was confirmed by using antisense c-fos and c-jun oligonucleotides. Studies conducted to understand the regulation of AP-1 and NF-kappaB activation by JNK MAPK revealed that both DXM and SP600125 inhibited IL-12p40 gene transcription by inhibiting the activation of AP-1 and NF-kappaB transcription factors as revealed by luciferase reporter and gel mobility shift assays. Taken together, our results suggest that DXM may inhibit IL-12p40 production in LPS-stimulated human monocytic cells by down-regulating the activation of JNK MAPK, the AP-1, and NF-kappaB transcription factors.

Gemfibrozil, a Lipid-lowering Drug, Inhibits the Induction of Nitric-oxide Synthase in Human Astrocytes*

PubMed Central

Pahan, Kalipada; Jana, Malabendu; Liu, Xiaojuan; Taylor, Bradley S.; Wood, Charles; Fischer, Susan M.

2007-01-01

Gemfibrozil, a lipid-lowering drug, inhibited cytokine-induced production of NO and the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astrocytes. Similar to gemfibrozil, clofibrate, another fibrate drug, also inhibited the expression of iNOS. Inhibition of human iNOS promoter-driven luciferase activity by gemfibrozil in cytokine-stimulated U373MG astroglial cells suggests that this compound inhibits the transcription of iNOS. Since gemfibrozil is known to activate peroxisome proliferator-activated receptor-α (PPAR-α), we investigated the role of PPAR-α in gemfibrozil-mediated inhibition of iNOS. Gemfibrozil induced peroxisome proliferator-responsive element (PPRE)-dependent luciferase activity, which was inhibited by the expression of ΔhPPAR-α, the dominant-negative mutant of human PPAR-α. However, ΔhPPAR-α was unable to abrogate gemfibrozil-mediated inhibition of iNOS suggesting that gemfibrozil inhibits iNOS independent of PPAR-α. The human iNOS promoter contains consensus sequences for the binding of transcription factors, including interferon-γ (IFN-γ) regulatory factor-1 (IRF-1) binding to interferon-stimulated responsive element (ISRE), signal transducer and activator of transcription (STAT) binding to γ-activation site (GAS), nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein β (C/EBPβ); therefore, we investigated the effect of gemfibrozil on the activation of these transcription factors. The combination of interleukin (IL)-1β and IFN-γ induced the activation of NF-κB, AP-1, C/EBPβ, and GAS but not that of ISRE, suggesting that IRF-1 may not be involved in cytokine-induced expression of iNOS in human astrocytes. Interestingly, gemfibrozil strongly inhibited the activation of NF-κB, AP-1, and C/EBPβ but not that of GAS in cytokine-stimulated astroglial cells. These results suggest that gemfibrozil inhibits the induction of iNOS probably by inhibiting the activation of NF-κB, AP-1, and C/EBPβ and that gemfibrozil, a prescribed drug for humans, may further find its therapeutic use in neuroinflammatory diseases. PMID:12244038

Fisetin, a flavonol, inhibits TH2-type cytokine production by activated human basophils.

PubMed

Higa, Shinji; Hirano, Toru; Kotani, Mayumi; Matsumoto, Motonobu; Fujita, Akihito; Suemura, Masaki; Kawase, Ichiro; Tanaka, Toshio

2003-06-01

Activation of mast cells and basophils through allergen stimulation releases chemical mediators and synthesizes cytokines. Among these cytokines, IL-4, IL-13, and IL-5 have major roles in allergic inflammation. We sought to determine the potency of flavonoids (astragalin, fisetin, kaempferol, myricetin, quercetin, and rutin) for the inhibition of cytokine expression and synthesis by human basophils. The inhibitory effect of flavonoids on cytokine expression by stimulated KU812 cells, a human basophilic cell line, and freshly purified peripheral blood basophils was measured by means of semiquantitative RT-PCR and ELISA assays. The effects of flavonoids on transcriptional activation of the nuclear factor of activated T cells were assessed by means of electrophoretic mobility shift assays. Fisetin suppressed the induction of IL-4, IL-13, and IL-5 mRNA expression by A23187-stimulated KU812 cells and basophils in response to cross-linkage of the IgE receptor. Fisetin reduced IL-4, IL-13, and IL-5 synthesis (inhibitory concentration of 50% [IC(50)] = 19.4, 17.7, and 17.4 micromol/L, respectively) but not IL-6 and IL-8 production by KU812 cells. In addition, fisetin inhibited IL-4 and IL-13 synthesis by anti-IgE antibody-stimulated human basophils (IC(50) = 5.1 and 6.2 micromol/L, respectively) and IL-4 synthesis by allergen-stimulated basophils from allergic patients (IC(50) = 4.8 micromol/L). Among the flavonoids examined, kaempferol and quercetin showed substantial inhibitory activities in cytokine expression but less so than those of fisetin. Fisetin inhibited nuclear localization of nuclear factor of activated T cells c2 by A23187-stimulated KU812 cells. These results provide evidence of a novel activity of the flavonoid fisetin that suppresses the expression of T(H)2-type cytokines (IL-4, IL-13, and IL-5) by basophils.

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor*

PubMed Central

Pucci, Mariangela; Pasquariello, Nicoletta; Battista, Natalia; Di Tommaso, Monia; Rapino, Cinzia; Fezza, Filomena; Zuccolo, Michela; Jourdain, Roland; Finazzi Agrò, Alessandro; Breton, Lionel; Maccarrone, Mauro

2012-01-01

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB1, CB2, and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μm) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB1-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μm). This CB1-dependent activity was fully abolished by the selective CB1 antagonist SR141716 or by RNA interference of the receptor. CB1 signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB1 activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor. PMID:22431736

Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli.

PubMed

Tanaka, H; Kaneko, T

1992-07-01

The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF). Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF. The effects of 5637- and recombinant hG-CSF administered via i.v. injection to rats showed similar response patterns of neutrophil counts in peripheral blood. From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities. The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay. After i.v. administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially. Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form. After s.c. administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.

Transforming growth factor-{alpha} enhances corneal epithelial cell migration by promoting EGFR recycling.

PubMed

McClintock, Jennifer L; Ceresa, Brian P

2010-07-01

PURPOSE. The goal of this study was to determine the molecular mechanism by which transforming growth factor-alpha (TGF-alpha) is a more potent activator of epidermal growth factor receptor (EGFR)-mediated corneal wound healing than epidermal growth factor (EGF). METHODS. Telomerase immortalized human corneal epithelial (hTCEpi) cells and primary human corneal epithelial cells were tested for their ability to migrate in response to EGF and TGF-alpha. In parallel, the endocytic trafficking of the EGFR in response to these same ligands was examined using indirect immunofluorescence, immunoblots, and radioligand binding. RESULTS. TGF-alpha, compared with EGF, is a more potent activator of corneal epithelial cell migration. Although both TGF-alpha and EGF were able to induce EGFR internalization and phosphorylation, only those receptors that were stimulated with EGF progressed to lysosomal degradation. EGFRs stimulated with TGF-alpha recycled back to the plasma membrane, where they could be reactivated with ligand. CONCLUSIONS. This study reveals that EGFR-mediated cell migration is limited by ligand-stimulated downregulation of the EGFR. This limitation can be overcome by treating cells with TGF-alpha because TGF-alpha stimulates EGFR endocytosis, but not degradation. After internalization of the TGF-alpha/EGFR complex, EGFR recycles back to the plasma membrane, where it can be restimulated. This sequence of events provides the receptor multiple opportunities for stimulation. Thus, stimulation with TGF-alpha prolongs EGFR signaling compared with EGF.

Endothelial cell stimulating angiogenesis factor.

PubMed

Weiss, J B; McLaughlin, B

1998-04-01

Endothelial cell stimulating angiogenesis factor (ESAF) is a small (> 1000 Da) dialysable non-peptide molecule with potent angiogenic activity. ESAF activates the major pro-matrix metalloproteinases and also uniquely reactivates the complex of these active enzymes with their tissue inhibitors resulting in both active enzyme and inhibitor. These actions may be pivotal in its role as an angiogenic factor. ESAF is primarily involved in angiogenic conditions where inflammatory cells are not evident such as foetal bone growth and electrically stimulated skeletal muscles and proliferative retinopathy. However, high levels also occur in actively growing human intracranial tumours but it is not noticeably elevated in rheumatoid arthritic synovial fluid. Its extreme potency and low molecular mass make its structural determination difficult. Possible therapeutic applications would be in the treatment of ischaemic ulcers, acceleration of fracture repair, infertility and more modestly in the correction of baldness. Analogues of ESAF could be of value in treating angiogenic diseases such as psoriasis and proliferative retinopathy.

Aciculatin Inhibits Granulocyte Colony-Stimulating Factor Production by Human Interleukin 1β-Stimulated Fibroblast-Like Synoviocytes

PubMed Central

Shih, Kao-Shang; Wang, Jyh-Horng; Wu, Yi-Wen; Teng, Che-Ming; Chen, Chien-Chih; Yang, Chia-Ron

2012-01-01

The expression of granulocyte colony-stimulating factor (G-CSF), the major regulator of neutrophil maturation, by human fibroblast-like synoviocytes (FLS) can be stimulated by the inflammatory cytokine interleukin-1β (IL-1β). G-CSF is known to contribute to the pathologic processes of destructive arthritis, but the induction mechanism remains unknown. The aims of this study were to identify the signaling pathways involved in IL-1β-stimulated G-CSF production and to determine whether this process was inhibited by aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), the major bioactive component of Chrysopogon aciculatus. IL-1β-induced cytokine expression was evaluated by measuring mRNA and protein levels by RT-PCR, ELISA, and Milliplex® assay. Whether aciculatin inhibited IL-1β-stimulated G-CSF expression, and if so, how, were evaluated using western blot assay, an electrophoretic mobility shift assay, and a reporter gene assay. Neutrophil differentiation was determined by Wright-Giemsa staining and flow cytometry. Aciculatin markedly inhibited G-CSF expression induced by IL-1β (10 ng/mL) in a concentration-dependent manner (1–10 µM). In clarifying the mechanisms involved, aciculatin was found to inhibit the IL-1β-induced activation of the IκB kinase (IKK)/IκB/nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways by suppressing the DNA binding activity of the transcription factors NF-κB and activator protein (AP)-1. Furthermore, aciculatin significantly inhibited the G-CSF-mediated phosphorylation of Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Akt and neutrophil differentiation from precursor cells. Our results show that aciculatin inhibits IL-1β-stimulated G-CSF expression and the subsequent neutrophil differentiation, suggesting that it might have therapeutic potential for inflammatory arthritis. PMID:22860122

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

PubMed Central

Auth, Marcus K.H.; Boost, Kim A.; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H.; Bechstein, Wolf-Otto; Blaheta, Roman A.

2005-01-01

AIM: Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. METHODS: Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. RESULTS: In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. CONCLUSION: Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors. PMID:15810072

Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique.

PubMed

Auth, Marcus-K H; Boost, Kim A; Leckel, Kerstin; Beecken, Wolf-Dietrich; Engl, Tobias; Jonas, Dietger; Oppermann, Elsie; Hilgard, Philip; Markus, Bernd H; Bechstein, Wolf-Otto; Blaheta, Roman A

2005-04-14

Clinical application of human hepatocytes (HC) is hampered by the progressive loss of growth and differentiation in vitro. The object of the study was to evaluate the effect of a biphasic culture technique on expression and activation of growth factor receptors and differentiation of human adult HC. Isolated HC were sequentially cultured in a hormone enriched differentiation medium (DM) containing nicotinamide, insulin, transferrin, selenium, and dexame-thasone or activation medium (AM) containing hepatocyte growth factor (HGF), epidermal growth factor (EGF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression, distribution and activation of the HC receptors (MET and EGFR) and the pattern of characteristic cytokeratin (CK) filaments were measured by fluorometry, confocal microscopy and Western blotting. In the biphasic culture system, HC underwent repeated cycles of activation (characterized by expression and activation of growth factor receptors) and re-differentiation (illustrated by distribution of typical filaments CK-18 but low or absent expression of CK-19). In AM increased expression of MET and EGFR was associated with receptor translocation into the cytoplasm and induction of atypical CK-19. In DM low expression of MET and EGFR was localized on the cell membrane and CK-19 was reduced. Receptor phosphorylation required embedding of HC in collagen type I gel. Control and reversible modulation of growth factor receptor activation of mature human HC can be accomplished in vitro, when defined signals from the extracellular matrix and sequential growth stimuli are provided. The biphasic technique helps overcome de-differentiation, which occurs during continuous stimulation by means of growth factors.

Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Kakeda, M; Yoneya, T; Tsumura, H; Ohashi, H; Yamaguchi, Y; Motoki, K; Sudo, T; Mori, K J

1996-09-01

The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.

Chemiluminometric Immuno-Analysis of Innate Immune Response against Repetitive Bacterial Stimulations for the Same Mammalian Cells

PubMed Central

Jeon, Jin-Woo; Cho, Il-Hoon; Ha, Un-Hwan; Seo, Sung-Kyu; Paek, Se-Hwan

2014-01-01

For monitoring of human cellular response to repetitive bacterial stimulations (e.g., Pseudomonas aeruginosa in a lysate form), we devised a chemiluminescent immuno-analytical system for toll-like receptor 1 (TLR1) as marker present on cell surfaces (e.g., A549). Upon stimulation, TLR1 recognizes pathogen-associated molecular patterns of the infectious agent and are then up-regulated via activation of the nuclear factor-κB (NF-κB) pathway. In this study, the receptor density was quantified by employing an antibody specific to the target receptor and by producing a chemiluminometric signal from an enzyme labeled to the binder. The activated status was then switched back to normal down-regulated stage, by changing the culture medium to one containing animal serum. The major factors affecting activation were the stimulation dose of the bacterial lysate, stimulation timing during starvation, and up- and down-regulation time intervals. Reiterative TLR regulation switching up to three times was not affected by either antibody remained after immunoassay or enzyme substrate (e.g., hydrogen peroxide) in solution. This immuno-analysis for TLRs could be unique to acquire accumulated response of the human cells to repeated stimulations and, therefore, can eventually apply to persistency testing of the cellular regulation in screening of anti-inflammatory substances. PMID:25109895

Doxorubicin resistance mediated by cytoplasmic macrophage colony-stimulating factor is associated with switch from apoptosis to autophagic cell death in MCF-7 breast cancer cells

PubMed Central

Zhang, Mengxia; Zhang, Hailiang; Tang, Fan; Wang, Yuhua; Mo, Zhongcheng; Lei, Xiaoyong

2016-01-01

Macrophage colony-stimulating factor is a vital factor in maintaining the biological function of monocyte–macrophage lineage. It is expressed in many tumor tissues and cancer cells. Recent findings indicate that macrophage colony-stimulating factor might contribute to chemoresistance, but the precise mechanisms are unclear. This study was to explore the effect of macrophage colony-stimulating factor on doxorubicin resistance in MCF-7 breast cancer cells and the possible mechanism. In the study, the human breast cancer cells, MCF-7, were transfected with macrophage colony-stimulating factor. We document that cytoplasmic macrophage colony-stimulating factor induces doxorubicin resistance and inhibits apoptosis in MCF-7 cells. Further studies demonstrated that cytoplasmic macrophage colony-stimulating factor-mediated apoptosis inhibition was dependent on the activation of PI3K/Akt/Survivin pathway. More importantly, we found that macrophage colony-stimulating factor-induced autophagic cell death in doxorubicin-treated MCF-7 cells. Taken together, we show for the first time that macrophage colony-stimulating factor-induced doxorubicin resistance is associated with the changes in cell death response with defective apoptosis and promotion of autophagic cell death. PMID:27439542

Cutting edge: IL-21 is a switch factor for the production of IgG1 and IgG3 by human B cells.

PubMed

Pène, Jérôme; Gauchat, Jean-François; Lécart, Sandrine; Drouet, Elodie; Guglielmi, Paul; Boulay, Vera; Delwail, Adriana; Foster, Don; Lecron, Jean-Claude; Yssel, Hans

2004-05-01

IL-21 is a cytokine that regulates the activation of T and NK cells and promotes the proliferation of B cells activated via CD40. In this study, we show that rIL-21 strongly induces the production of all IgG isotypes by purified CD19(+) human spleen or peripheral blood B cells stimulated with anti-CD40 mAb. Moreover, it was found to specifically induce the production of IgG(1) and IgG(3) by CD40-activated CD19(+)CD27(-) naive human B cells. Although stimulation of CD19(+) B cells via CD40 alone induced gamma 1 and gamma 3 germline transcripts, as well as the expression of activation-induced cytidine deaminase, only stimulation with both anti-CD40 mAb and rIL-21 resulted in the production of S gamma/S mu switch circular DNA. These results show that IL-21, in addition to promoting growth and differentiation of committed B cells, is a specific switch factor for the production of IgG(1) and IgG(3).

Novel mechanism and factor for regulation by HIV-1 Tat.

PubMed Central

Zhou, Q; Sharp, P A

1995-01-01

Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression. Images PMID:7835343

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Hae-Ryung, E-mail: heaven@umich.edu; Loch-Caruso, Rita

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20 μM BDE-47more » for 24 h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20 μM BDE-47 for 24 h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. - Highlights: • BDE-47 stimulated ARE reporter activity and GSH production. • BDE-47 resulted in differential expression of redox-sensitive genes. • Nrf2 inducers upregulated Nrf2-mediated oxidative stress responses. • Nrf2 inducers reduced BDE-47-stimulated IL-6 release and NF-κB activity.« less

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

PubMed

Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

The Estuary.

ERIC Educational Resources Information Center

Fortner, Rosanne; Bainer, Deborah L.

1983-01-01

Provides an OEAGLS (Oceanic Education Activities for Great Lakes Schools) activity showing how biological sampling can be taught using acquatic examples in a "dry lab." The activity, requiring use of mathematical/observational skills, simulates a plankton sample and stimulates discussions of how environmental/human factors can cause…

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

PubMed

Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Green tea polyphenol epigallocatechin-3-gallate inhibits advanced glycation end product-induced expression of tumor necrosis factor-alpha and matrix metalloproteinase-13 in human chondrocytes.

PubMed

Rasheed, Zafar; Anbazhagan, Arivarasu N; Akhtar, Nahid; Ramamurthy, Sangeetha; Voss, Frank R; Haqqi, Tariq M

2009-01-01

The major risk factor for osteoarthritis (OA) is aging, but the mechanisms underlying this risk are only partly understood. Age-related accumulation of advanced glycation end products (AGEs) can activate chondrocytes and induce the production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In the present study, we examined the effect of epigallocatechin-3-gallate (EGCG) on AGE-modified-BSA (AGE-BSA)-induced activation and production of TNFalpha and MMP-13 in human OA chondrocytes. Human chondrocytes were derived from OA cartilage by enzymatic digestion and stimulated with in vitro-generated AGE-BSA. Gene expression of TNFalpha and MMP-13 was measured by quantitative RT-PCR. TNFalpha protein in culture medium was determined using cytokine-specific ELISA. Western immunoblotting was used to analyze the MMP-13 production in the culture medium, phosphorylation of mitogen-activated protein kinases (MAPKs), and the activation of NF-kappaB. DNA binding activity of NF-kappaB p65 was determined using a highly sensitive and specific ELISA. IkappaB kinase (IKK) activity was determined using an in vitro kinase activity assay. MMP-13 activity in the culture medium was assayed by gelatin zymography. EGCG significantly decreased AGE-stimulated gene expression and production of TNFalpha and MMP-13 in human chondrocytes. The inhibitory effect of EGCG on the AGE-BSA-induced expression of TNFalpha and MMP-13 was mediated at least in part via suppression of p38-MAPK and JNK activation. In addition, EGCG inhibited the phosphorylating activity of IKKbeta kinase in an in vitro activity assay and EGCG inhibited the AGE-mediated activation and DNA binding activity of NF-kappaB by suppressing the degradation of its inhibitory protein IkappaBalpha in the cytoplasm. These novel pharmacological actions of EGCG on AGE-BSA-stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG-derived compounds may inhibit cartilage degradation by suppressing AGE-mediated activation and the catabolic response in human chondrocytes.

Increased C3 production in human monocytes after stimulation with Candida albicans is suppressed by granulocyte-macrophage colony-stimulating factor.

PubMed Central

Høgåsen, A K; Abrahamsen, T G

1993-01-01

Activation of the complement system is an important part of host resistance against fungal infections. When human monocytes, cultured for 2 days or more, were treated in vitro with Candida albicans for 24 h, an enhancement of their biosynthesis of the complement components C3 and factor B was found. However, when C. albicans was administered to freshly isolated monocytes, a consistent stimulation of factor B biosynthesis occurred, while the C3 production was increased in about 50% of the donors. C. albicans also induced the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the cultured cells, apparently in larger amounts in the donors in whom no stimulation of C3 production was found. An antibody to GM-CSF administered with the yeast at the initiation of the monocyte culture caused an increase in the C3 production. Furthermore, when monocytes were treated with recombinant human GM-CSF either at the same time as or 4 days prior to the addition of C. albicans, the increase in C3 production was suppressed or neutralized, while factor B biosynthesis was unaffected. Taken together, these results indicate that monocytes respond to C. albicans with an increased production of complement factors. This may be an important mechanism both for opsonization of the fungus and for initiation of an inflammatory reaction. At an inflammatory site, this complement response may be suppressed by locally produced GM-CSF. PMID:8478067

Does granulocyte colony-stimulating factor ameliorate the proinflammatory response in human meningococcal septic shock?

PubMed

Rojahn, Astrid; Brusletto, Berit; Øvstebø, Reidun; Haug, Kari B F; Kierulf, Peter; Brandtzaeg, Petter

2008-09-01

To test the hypothesis that granulocyte colony-stimulating factor acts cooperatively with interleukin-10 in down-regulating monocyte function in severe meningococcal septic shock. 1) We quantified the plasma levels of granulocyte colony-stimulating factor, interleukin-10, Neisseria meningitidis lipopolysaccharide and the number of N. meningitidis DNA copies in 28 patients with systemic meningococcal disease. 2) We studied the inhibitory effect of recombinant human granulocyte colony-stimulating factor on normal human monocytes stimulated with purified meningococcal lipopolysaccaride. 3) We monitored the inhibitory effects of endogenously produced granulocyte colony-stimulating factor and interleukin-10 in meningococcal shock plasmas on monocytes. Comparative, experimental study. University Hospital and laboratory. Twenty-eight patients with systemic meningococcal disease, 13 with persistent shock, 7 died, and 15 without shock. The median levels of granulocyte colony-stimulating factor in shock and nonshock patients were 1.7 x 10(6) and 8.1 x 10(2) pg/mL; interleukin-10, 2.1 x 10(4) and 4 x 10(1) pg/mL; number of N. meningitidis DNA copies, 2.9 x 10(7) and <10(3)/mL; and lipopolysaccharide, 105 and <0.04 endotoxin units/mL, respectively. The plasma levels of granulocyte colony-stimulating factor were reduced by 50% within 4 to 6 hrs after initiation of antibiotic treatment. In model experiments with lipopolysaccharide-stimulated human monocytes, recombinant human granulocyte colony-stimulating factor and interleukin-10 reduced the release of tumor necrosis factor-alpha by mean 30% and 92%, respectively. When plasmas from three shock patients were depleted of native granulocyte colony-stimulating factor or interleukin-10 by immunoprecipitation, no increase in tumor necrosis factor-alpha release occurred after removal of granulocyte colony-stimulating factor, whereas removal of interleukin-10 increased the tumor necrosis factor-alpha release eight-fold. Although granulocyte colony-stimulating factor in plasma increases by five orders of magnitude in patients with meningococcal shock, the anti-inflammatory effect on patients' monocytes is uncertain.

Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling.

PubMed

Niwano, Takao; Terazawa, Shuko; Nakajima, Hiroaki; Wakabayashi, Yuki; Imokawa, Genji

2015-06-01

Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes. These results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A). Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

PubMed

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

Lactoferrin from Camelus dromedarius Inhibits Nuclear Transcription Factor-kappa B Activation, Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Stimulated Human Chondrocytes

PubMed Central

Rasheed, Naila; Alghasham, Abdullah; Rasheed, Zafar

2016-01-01

Background: Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. Nuclear factor-kappa B (NF)-κB is a major cellular event in OA and its activation by interleukin-1β (IL-1β) plays a critical role in cartilage breakdown in these patients. Objective: In this study, we examined the effect of lactoferrin on NF-κB activation, cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in stimulated human articular chondrocytes. Materials and Methods: Human chondrocytes were derived from OA articular cartilage and treated with camel lactoferrin and then stimulated with IL-1β. Gene expression was determined by TaqMan assays and protein expression was studied by Western immunoblotting. NF-κB activity and PGE2 levels were determined by ELISA based assays. NF-κB activity was also determined by treatment of chondrocytes with NF-κB specific inhibitor Bay 11–7082. Results: Lactoferrin inhibited IL-1β-induced activation and nuclear translocation of NF-κB p65 in human OA chondrocytes. Lactoferrin also inhibited mRNA/protein expression of COX-2 and production of PGE2. Moreover, Bay 11–7082 also inhibited IL-1β-induced expression of COX-2 and production of PGE2. The inhibitory effect of lactoferrin on the IL-1β induced expression of COX-2 or production of PGE2 was mediated at least in part via suppression of NF-κB activation. Conclusions: Our data determine camel lactoferrin as a novel inhibitor of IL-1β-induced activation of NF-κB signaling events and production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expressions. These results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA and other degenerative/inflammatory diseases. SUMMARY Lactoferrin shows anti-arthritic activity in IL-1β stimulated primary human chondrocytes.Lactoferrin inhibits IL-1β-induced NF-κB activation.Lactoferrin inhibits production of cartilage degrading PGE2 via inhibition of COX-2 expression. Abbreviations Used: OA: Osteoarthritis IL-1β: Interleukin-1 beta NF-κB: Nuclear factor-kappa B COX-2: cyclooxygenase-2 PGE2: prostaglandin E2 PMID:27034605

Lactoferrin from Camelus dromedarius Inhibits Nuclear Transcription Factor-kappa B Activation, Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Stimulated Human Chondrocytes.

PubMed

Rasheed, Naila; Alghasham, Abdullah; Rasheed, Zafar

2016-01-01

Osteoarthritis (OA) is a progressive joint disorder, which remains the leading cause of chronic disability in aged people. Nuclear factor-kappa B (NF)-κB is a major cellular event in OA and its activation by interleukin-1β (IL-1β) plays a critical role in cartilage breakdown in these patients. In this study, we examined the effect of lactoferrin on NF-κB activation, cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production in stimulated human articular chondrocytes. Human chondrocytes were derived from OA articular cartilage and treated with camel lactoferrin and then stimulated with IL-1β. Gene expression was determined by TaqMan assays and protein expression was studied by Western immunoblotting. NF-κB activity and PGE2 levels were determined by ELISA based assays. NF-κB activity was also determined by treatment of chondrocytes with NF-κB specific inhibitor Bay 11-7082. Lactoferrin inhibited IL-1β-induced activation and nuclear translocation of NF-κB p65 in human OA chondrocytes. Lactoferrin also inhibited mRNA/protein expression of COX-2 and production of PGE2. Moreover, Bay 11-7082 also inhibited IL-1β-induced expression of COX-2 and production of PGE2. The inhibitory effect of lactoferrin on the IL-1β induced expression of COX-2 or production of PGE2 was mediated at least in part via suppression of NF-κB activation. Our data determine camel lactoferrin as a novel inhibitor of IL-1β-induced activation of NF-κB signaling events and production of cartilage-degrading molecule PGE2 via inhibition of COX-2 expressions. These results may have important implications for the development of novel therapeutic strategies for the prevention/treatment of OA and other degenerative/inflammatory diseases. Lactoferrin shows anti-arthritic activity in IL-1β stimulated primary human chondrocytes.Lactoferrin inhibits IL-1β-induced NF-κB activation.Lactoferrin inhibits production of cartilage degrading PGE2 via inhibition of COX-2 expression. Abbreviations Used: OA: Osteoarthritis IL-1β: Interleukin-1 beta NF-κB: Nuclear factor-kappa B COX-2: cyclooxygenase-2 PGE2: prostaglandin E2.

Identification of Poly(ADP-Ribose) Polymerase as a Transcriptional Coactivator of the Human T-Cell Leukemia Virus Type 1 Tax Protein

PubMed Central

Anderson, Mark G.; Scoggin, Kirsten E. S.; Simbulan-Rosenthal, Cynthia M.; Steadman, Jennifer A.

2000-01-01

Human T-cell leukemia virus type 1 (HTLV-1) encodes a transcriptional activator, Tax, whose activity is believed to contribute significantly to cellular transformation. Tax stimulates transcription from the proviral promoter as well as from promoters for a variety of cellular genes. The mechanism through which Tax communicates to the general transcription factors and RNA polymerase II has not been completely determined. We investigated whether Tax could function directly through the general transcription factors and RNA polymerase II or if other intermediary factors or coactivators were required. Our results show that a system consisting of purified recombinant TFIIA, TFIIB, TFIIE, TFIIF, CREB, and Tax, along with highly purified RNA polymerase II, affinity-purified epitope-tagged TFIID, and semipurified TFIIH, supports basal transcription of the HTLV-1 promoter but is not responsive to Tax. Two additional activities were required for Tax to stimulate transcription. We demonstrate that one of these activities is poly(ADP-ribose) polymerase (PARP), a molecule that has been previously identified to be the transcriptional coactivator PC1. PARP functions as a coactivator in our assays at molar concentrations approximately equal to those of the DNA and equal to or less than those of the transcription factors in the assay. We further demonstrate that PARP stimulates Tax-activated transcription in vivo, demonstrating that this biochemical approach has functionally identified a novel target for the retroviral transcriptional activator Tax. PMID:10666246

Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

PubMed

Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

2011-04-01

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Yoneya, T; Kakeda, M; Tsumura, H; Ohashi, H; Mori, K J

1997-04-01

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

Induction of cyclooxygenase-2 expression by prostaglandin E2 stimulation of the prostanoid EP4 receptor via coupling to Gαi and transactivation of the epidermal growth factor receptor in HCA-7 human colon cancer cells.

PubMed

Yoshida, Kenji; Fujino, Hiromichi; Otake, Sho; Seira, Naofumi; Regan, John W; Murayama, Toshihiko

2013-10-15

Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gαi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis. © 2013 Elsevier B.V. All rights reserved.

Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Husmann, Knut, E-mail: khusmann@research.balgrist.ch; Ducommun, Pascal; Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich

2015-09-04

The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS andmore » 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.« less

Leukemia inhibitory factor: a novel bone-active cytokine.

PubMed

Reid, L R; Lowe, C; Cornish, J; Skinner, S J; Hilton, D J; Willson, T A; Gearing, D P; Martin, T J

1990-03-01

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

Mangiferin inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 expression and cellular invasion by suppressing nuclear factor-κB activity

PubMed Central

Dilshara, Matharage Gayani; Kang, Chang-Hee; Choi, Yung Hyun; Kim, Gi-Young

2015-01-01

We investigated the effects of mangiferin on the expression and activity of metalloproteinase (MMP)-9 and the invasion of tumor necrosis factor (TNF)-α-stimulated human LNCaP prostate carcinoma cells. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that mangiferin significantly reversed TNF-α-induced mRNA and protein expression of MMP-9 expression. Zymography data confirmed that stimulation of cells with TNF-α significantly increased MMP-9 activity. However, mangiferin substantially reduced the TNF-α-induced activity of MMP-9. Additionally, a matrigel invasion assay showed that mangiferin significantly reduced TNF-α-induced invasion of LNCaP cells. Compared to untreated controls, TNF-α-stimulated LNCaP cells showed a significant increase in nuclear factor-κB (NF-κB) luciferase activity. However, mangiferin treatment markedly decreased TNF-α-induced NF-κB luciferase activity. Furthermore, mangiferin suppressed nuclear translocation of the NF-κB subunits p65 and p50. Collectively, our results indicate that mangiferin is a potential anti-invasive agent that acts by suppressing NF-κB-mediated MMP-9 expression. [BMB Reports 2015; 48(10): 559-564] PMID:25739392

Mangiferin inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 expression and cellular invasion by suppressing nuclear factor-κB activity.

PubMed

Dilshara, Matharage Gayani; Kang, Chang-Hee; Choi, Yung Hyun; Kim, Gi-Young

2015-10-01

We investigated the effects of mangiferin on the expression and activity of metalloproteinase (MMP)-9 and the invasion of tumor necrosis factor (TNF)-α-stimulated human LNCaP prostate carcinoma cells. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis showed that mangiferin significantly reversed TNF-α-induced mRNA and protein expression of MMP-9 expression. Zymography data confirmed that stimulation of cells with TNF-α significantly increased MMP-9 activity. However, mangiferin substantially reduced the TNF-α-induced activity of MMP-9. Additionally, a matrigel invasion assay showed that mangiferin significantly reduced TNF-α-induced invasion of LNCaP cells. Compared to untreated controls, TNF-α-stimulated LNCaP cells showed a significant increase in nuclear factor-κB (NF-κB) luciferase activity. However, mangiferin treatment markedly decreased TNF-α-induced NF-κB luciferase activity. Furthermore, mangiferin suppressed nuclear translocation of the NF-κB subunits p65 and p50. Collectively, our results indicate that mangiferin is a potential anti-invasive agent that acts by suppressing NF-κB-mediated MMP-9 expression.

Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts.

PubMed

Heo, Jung Sun; Lee, Seung-Youp; Lee, Jeong-Chae

2010-11-01

Wnt/β-catenin signaling has been known to influence bone formation and homeostasis. In this study, we investigated the canonical Wnt signaling regulation of osteogenic differentiation from periodontal ligament (PDL) fibroblasts. Stimulating PDL fibroblasts with lithium chloride (LiCl), a canonical Wnt activator, significantly increased mineralized nodule and alkaline phosphatase (ALP) activity in a time- and dose-dependent manner. LiCl up-regulated protein expression of osteogenic transcription factors, including the runt-related gene 2, Msx2, and Osterix 2, in the PDL fibroblasts. Treatment of these cells with LiCl also increased the mRNA levels of ALP, FosB, and Fra1 in a dose-dependent manner. Blockage of canonical Wnt signaling by treating the cells with DKK1 inhibited Wnt1-stimulated mRNA expression of these osteogenic factors. Furthermore, pretreatment with DKK1 reduced the ALP activity and matrix mineralization stimulated by Wnt1. Collectively, these results suggest that canonical Wnt signaling leads to the differentiation of PDL fibroblasts into osteogenic lineage with the attendant stimulation of osteogenic transcription factors.

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing

PubMed Central

Koob, Thomas J; Rennert, Robert; Zabek, Nicole; Massee, Michelle; Lim, Jeremy J; Temenoff, Johnna S; Li, William W; Gurtner, Geoffrey

2013-01-01

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme-linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, transforming growth factor α (TGFα), TGFβ1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony-stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL-4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications. PMID:23902526

Lactoferrin release and interleukin-1, interleukin-6, and tumor necrosis factor production by human polymorphonuclear cells stimulated by various lipopolysaccharides: relationship to growth inhibition of Candida albicans.

PubMed

Palma, C; Cassone, A; Serbousek, D; Pearson, C A; Djeu, J Y

1992-11-01

Lipopolysaccharides (LPSs) from Escherichia coli, Serratia marcescens, and Salmonella typhimurium, at doses from 1 to 100 ng/ml, strongly enhanced growth inhibition of Candida albicans by human polymorphonuclear leukocytes (PMN) in vitro. Flow cytometry analysis demonstrated that LPS markedly augmented phagocytosis of Candida cells by increasing the number of yeasts ingested per neutrophil as well as the number of neutrophils capable of ingesting fungal cells. LPS activation caused augmented release of lactoferrin, an iron-binding protein which itself could inhibit the growth of C. albicans in vitro. Antibodies against lactoferrin effectively and specifically reduced the anti-C. albicans activity of both LPS-stimulated and unstimulated PMN. Northern (RNA blot) analysis showed enhanced production of mRNAs for interleukin-1 beta, tumor necrosis factor alpha, and interleukin-6 and in neutrophils within 1 h of stimulation with LPS. The cytokines were also detected in the supernatant of the activated PMN, and their synthesis was prevented by pretreatment of LPS-stimulated PMN with protein synthesis inhibitors, such as emetine and cycloheximide. These inhibitors, however, did not block either lactoferrin release or the anti-Candida activity of LPS-stimulated PMN. These results demonstrate the ability of various bacterial LPSs to augment neutrophil function against C. albicans and suggest that the release of a candidastatic, iron-binding protein, lactoferrin, may contribute to the antifungal effect of PMN. Moreover, the ability to produce cytokines upon stimulation by ubiquitous microbial products such as the endotoxins points to an extraphagocytic, immunomodulatory role of PMN during infection.

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

PubMed Central

Tamada, Taro; Honjo, Eijiro; Maeda, Yoshitake; Okamoto, Tomoyuki; Ishibashi, Matsujiro; Tokunaga, Masao; Kuroki, Ryota

2006-01-01

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 Å resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses. PMID:16492764

Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes.

PubMed

Xu, Y; Greenstock, C L; Trivedi, A; Mitchel, R E

1996-05-01

Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell.

Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

EPA Science Inventory

Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase.

PubMed

Kaufmann, Isabelle; Martin, Georges; Friedlein, Arno; Langen, Hanno; Keller, Walter

2004-02-11

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

PubMed Central

Kaufmann, Isabelle; Martin, Georges; Friedlein, Arno; Langen, Hanno; Keller, Walter

2004-01-01

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity. PMID:14749727

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

PubMed

Watanabe, M; Fukamachi, H; Uzumaki, H; Kabaya, K; Tsumura, H; Ishikawa, M; Matsuki, S; Kusaka, M

1991-05-15

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

Pituitary adenylate cyclase-activating polypeptide is a potent inhibitor of the growth of light chain-secreting human multiple myeloma cells.

PubMed

Li, Min; Cortez, Shirley; Nakamachi, Tomoya; Batuman, Vecihi; Arimura, Akira

2006-09-01

Multiple myeloma represents a malignant proliferation of plasma cells in the bone marrow, which often overproduces immunoglobulin light chains. We have shown previously that pituitary adenylate cyclase-activating polypeptide (PACAP) markedly suppresses the release of proinflammatory cytokines from light chain-stimulated human renal proximal tubule epithelial cells and prevents the resulting tubule cell injury. In this study, we have shown that PACAP suppresses the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. The addition of PACAP suppressed light chain-producing myeloma cell-stimulated interleukin 6 (IL-6) secretion by the bone marrow stromal cells (BMSCs). A specific antagonist to either the human PACAP-specific receptor or the vasoactive intestinal peptide receptor attenuated the suppressive effect of PACAP on IL-6 production in the adhesion of human multiple myeloma cells to BMSCs. The secretion of IL-6 by BMSCs was completely inhibited by 10(-9) mol/L PACAP, which also attenuated the phosphorylation of both p42/44 and p38 mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappaB (NF-kappaB) activation in response to the adhesion of multiple myeloma cells to BMSCs, whereas the inhibition of p42/44 MAPK signaling attenuated PACAP action. The signaling cascades involved in the inhibitory effect of PACAP on IL-6-mediated paracrine stimulation of light chain-secreting myeloma cell growth was mediated through the suppression of p38 MAPK as well as modulation of activation of transcription factor NF-kappaB. These findings suggest that PACAP may be a new antitumor agent that directly suppresses light chain-secreting myeloma cell growth and indirectly affects tumor cell growth by modifying the bone marrow milieu of the multiple myeloma.

Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression

PubMed Central

1990-01-01

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1-long terminal repeat (HIV-1-LTR) CAT constructs transfected into monocyte/macrophage-like cell lines but not a T cell line. This effect appears to be mediated through the induction of nuclear factor kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrate that LPS induces a DNA binding activity indistinguishable from NF-kappa B in U937 and THP-1 cells. LPS is also shown to dramatically increase HIV-1 production from a chronically infected monocyte/macrophage-like cloned cell line, U1, which produces very low levels of HIV-1 at baseline. The stimulation of viral production from this cell line occurs only if these cells are treated with granulocyte/macrophage colony-stimulating factor (GM-CSF) before treatment with LPS. This stimulation of HIV-1 production is correlated with an increase in the level of HIV-1 RNA and and activation of NF- kappa B. LPS is not able to induce HIV-1 production in a cloned T cell line. The effect of LPS on HIV-1 replication occurs at picogram per milliliter concentrations and may be clinically significant in understanding the variability of the natural history of HIV-1 infection. PMID:2193097

Tissue factor-expressing monocytes inhibit fibrinolysis through a TAFI-mediated mechanism, and make clots resistant to heparins

PubMed Central

Semeraro, Fabrizio; Ammollo, Concetta T.; Semeraro, Nicola; Colucci, Mario

2009-01-01

Background Thrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro. Design and Methods Tissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator. Results LPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/μL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (~50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity. Conclusions Our data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor-mediated enhancement of thrombin activatable fibrinolysis inhibitor activation and make clots resistant to the profibrinolytic activity of heparins, thus providing an additional mechanism whereby tissue factor-expressing monocytes/macrophages may favor fibrin accumulation and diminish the antithrombotic efficacy of heparins. PMID:19377079

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

PubMed

Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

2002-09-04

The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Dexamethasone and interleukin-1 potently synergize to stimulate the production of granulocyte colony-stimulating factor in differentiated THP-1 cells.

PubMed

Wang, Y; Zhang, J J; Lei, K Y; Pike, J W

1997-10-29

The human monocytic leukemic cell line, THP-1, which differentiates toward macrophages in response to phorbol 12-myristate 13-acetate (PMA) was investigated for its ability to produce granulocyte colony-stimulating factor (G-CSF). G-CSF protein was neither produced during PMA-induced differentiation nor in response to dexamethasone (Dex) alone. However, when combined, PMA and Dex synergistically stimulated THP-1 cells to produce G-CSF. The synergistic interaction between PMA and Dex on G-CSF production appeared to be mediated through the production of interleukin-1 (IL-1) since neutralization of IL-1 activity completely inhibited G-CSF production. Further experiments demonstrated that in THP-1 cells pretreated with PMA, Dex potently synergized with IL-1 to stimulate G-CSF production.

Differential growth factor control of bone formation through osteoprogenitor differentiation.

PubMed

Chaudhary, L R; Hofmeister, A M; Hruska, K A

2004-03-01

The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.

Synergistic effect of concanavalin A and Bu-WSA on DNA synthesis in human peripheral blood lymphocytes.

PubMed

Nitta, T; Okumura, S; Nakano, M

1985-02-01

Butanol-extracted water soluble adjuvant (Bu-WSA) obtained from Bacterionema matruchotii was not mitogenic for human peripheral blood mononuclear cells (PBM) but was capable of enhancing (3H) thymidine uptake of T cells stimulated by concanavalin A (Con A) in the presence of B cells or macrophages (M phi) in vitro. The mechanisms of the synergy of Con A and Bu-WSA were studied by using separated cell populations from PBM. Both subfractioned OKT4+ and OKT8+ cells were responsive to co-stimulation by Con A and Bu-WSA in the presence of an accessory cell population. Allogeneic B cells and M phi as well as autologous cells had helper function as accessory cells. Heavy irradiation with gamma-rays did not affect the function of the accessory cells, but previous treatment of B cells with anti-Ig serum plus complement (C) or treatment of M phi with anti-M phi serum plus C deprived them of their function. The treatment of accessory cells with anti-HLA-DR serum, regardless of the presence or absence of C, resulted in loss of their helper function. Cultures in Marbrook-type vessels showed that a mixed cell population of T cells and accessory cells in the lower chamber produced some active factor(s) after co-stimulation with Con A and Bu-WSA, and by passing through the membrane filter separating the chambers, the factor(s) enhanced the proliferation of the Con A-activated T cell population in the upper chamber. The factor(s) was presumed to be interleukin 2 (IL 2), because it supported the growth of IL 2-dependent CTLL cells. These results indicate that the synergy of Con A and Bu-WSA on the proliferative response of human PBM is due to the elevation of growth factor production from T cells stimulated by those mitogens.

Zyxin regulates migration of renal epithelial cells through activation of hepatocyte nuclear factor-1β.

PubMed

Choi, Yun-Hee; McNally, Brian T; Igarashi, Peter

2013-07-01

Hepatocyte nuclear factor-1β (HNF-1β) is an epithelial tissue-specific transcription factor that regulates gene expression in the kidney, liver, pancreas, intestine, and other organs. Mutations of HNF-1β in humans produce renal cysts and congenital kidney anomalies. Here, we identify the LIM-domain protein zyxin as a novel binding partner of HNF-1β in renal epithelial cells. Zyxin shuttles to the nucleus where it colocalizes with HNF-1β. Immunoprecipitation of zyxin in leptomycin B-treated cells results in coprecipitation of HNF-1β. The protein interaction requires the second LIM domain of zyxin and two distinct domains of HNF-1β. Overexpression of zyxin stimulates the transcriptional activity of HNF-1β, whereas small interfering RNA silencing of zyxin inhibits HNF-1β-dependent transcription. Epidermal growth factor (EGF) induces translocation of zyxin into the nucleus and stimulates HNF-1β-dependent promoter activity. The EGF-mediated nuclear translocation of zyxin requires activation of Akt. Expression of dominant-negative mutant HNF-1β, knockdown of zyxin, or inhibition of Akt inhibits EGF-stimulated cell migration. These findings reveal a novel pathway by which extracellular signals are transmitted to the nucleus to regulate the activity of a transcription factor that is essential for renal epithelial differentiation.

21 CFR 340.10 - Stimulant active ingredient.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 5 2014-04-01 2014-04-01 false Stimulant active ingredient. 340.10 Section 340.10... FOR HUMAN USE STIMULANT DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Active Ingredient § 340.10 Stimulant active ingredient. The active ingredient of the product consists of caffeine when used within the...

21 CFR 340.10 - Stimulant active ingredient.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 5 2012-04-01 2012-04-01 false Stimulant active ingredient. 340.10 Section 340.10... FOR HUMAN USE STIMULANT DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Active Ingredient § 340.10 Stimulant active ingredient. The active ingredient of the product consists of caffeine when used within the...

21 CFR 340.10 - Stimulant active ingredient.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 5 2011-04-01 2011-04-01 false Stimulant active ingredient. 340.10 Section 340.10... FOR HUMAN USE STIMULANT DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Active Ingredient § 340.10 Stimulant active ingredient. The active ingredient of the product consists of caffeine when used within the...

21 CFR 340.10 - Stimulant active ingredient.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 5 2013-04-01 2013-04-01 false Stimulant active ingredient. 340.10 Section 340.10... FOR HUMAN USE STIMULANT DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Active Ingredient § 340.10 Stimulant active ingredient. The active ingredient of the product consists of caffeine when used within the...

Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.

PubMed

McDermott, Catherine; Chess-Williams, Russ; Grant, Gary D; Perkins, Anthony V; McFarland, Amelia J; Davey, Andrew K; Anoopkumar-Dukie, Shailendra

2012-03-01

We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Activation of AMPK stimulates heme oxygenase-1 gene expression and human endothelial cell survival

PubMed Central

Liu, Xiao-ming; Peyton, Kelly J.; Shebib, Ahmad R.; Wang, Hong; Korthuis, Ronald J.

2011-01-01

The present study determined whether AMP-activated protein kinase (AMPK) regulates heme oxygenase (HO)-1 gene expression in endothelial cells (ECs) and if HO-1 contributes to the biological actions of this kinase. Treatment of human ECs with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) stimulated a concentration- and time-dependent increase in HO-1 protein and mRNA expression that was associated with a prominent increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) protein. Induction of HO-1 was also observed in rat carotid arteries after the in vivo application of AICAR. Induction of HO-1 by AICAR was blocked by the AMPK inhibitor compound C, the adenosine kinase inhibitor 5′-iodotubercidin, and by silencing AMPK-α1/2 and was mimicked by the AMPK activator A-769662 and by infecting ECs with an adenovirus expressing constitutively active AMPK-α1. AICAR also induced a significant rise in HO-1 promoter activity that was abolished by mutating the antioxidant responsive elements of the HO-1 promoter or by the overexpression of dominant negative Nrf2. Finally, activation of AMPK inhibited cytokine-mediated EC death, and this was prevented by the HO inhibitor tin protoporphyrin-IX or by silencing HO-1 expression. In conclusion, AMPK stimulates HO-1 gene expression in human ECs via the Nrf2/antioxidant responsive element signaling pathway. The induction of HO-1 mediates the antiapoptotic effect of AMPK, and this may provide an important adaptive response to preserve EC viability during periods of metabolic stress. PMID:21037234

Neurotensin is an autocrine trophic factor stimulated by androgen withdrawal in human prostate cancer.

PubMed Central

Sehgal, I; Powers, S; Huntley, B; Powis, G; Pittelkow, M; Maihle, N J

1994-01-01

After therapeutic hormone deprivation, prostate cancer cells often develop androgen-insensitive growth through mechanisms thus far undefined. Neuropeptides have been previously implicated as growth factors in some prostate cancers. Here, we demonstrate that androgen-sensitive LNCaP human prostate cancer cells produce and secrete neurotensin following androgen withdrawal. We show that while LNCaP cells express the neurotensin receptor, only androgen-deprived cells exhibit a growth response to exogenous neurotensin. We further demonstrate that androgen-stimulated cells may be refractory to exogenous neurotensin due to androgen induction of a metalloprotease active toward neurotensin. Thus, prostate cancer cells deprived of androgen develop an alternative autocrine growth mechanism involving neurotensin. Images PMID:8197117

Stimulation of interleukin-13 expression by human T-cell leukemia virus type 1 oncoprotein Tax via a dually active promoter element responsive to NF-kappaB and NFAT.

PubMed

Silbermann, Katrin; Schneider, Grit; Grassmann, Ralph

2008-11-01

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein transforms human lymphocytes and is critical for the pathogenesis of HTLV-1-induced adult T-cell leukaemia. In HTLV-transformed cells, Tax upregulates interleukin (IL)-13, a cytokine with proliferative and anti-apoptotic functions that is linked to leukaemogenesis. Tax-stimulated IL-13 is thought to result in autocrine stimulation of HTLV-infected cells and thus may be relevant to their growth. The causal transactivation of the IL-13 promoter by Tax is predominantly dependent on a nuclear factor of activated T cells (NFAT)-binding P element. Here, it was shown that the isolated IL-13 Tax-responsive element (IL13TaxRE) was sufficient to mediate IL-13 transactivation by Tax and NFAT1. However, cyclosporin A, a specific NFAT inhibitor, revealed that Tax transactivation of IL13TaxRE or wild-type IL-13 promoter was independent of NFAT and that NFAT did not contribute to IL-13 upregulation in HTLV-transformed cells. By contrast, Tax stimulation was repressible by an efficient nuclear factor (NF)-kappaB inhibitor (IkBaDN), indicating the requirement for NF-kappaB. The capacity of NF-kappaB to stimulate IL13TaxRE was demonstrated by a strong response to NF-kappaB in reporter assays and by direct binding of NF-kappaB to IL13TaxRE. Thus, IL13TaxRE in the IL-13 promoter represents a dually active promoter element responsive to NF-kappaB and NFAT. Together, these results indicate that Tax causes IL-13 upregulation in HTLV-1-infected cells via NF-kappaB.

Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

PubMed

Stegajev, Vasili; Kouri, Vesa-Petteri; Salem, Abdelhakim; Rozov, Stanislav; Stark, Holger; Nordström, Dan C E; Konttinen, Yrjö T

2014-12-01

Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells

PubMed Central

Garonna, Elena; Botham, Kathleen M.; Birdsey, Graeme M.; Randi, Anna M.; Gonzalez-Perez, Ruben R.; Wheeler-Jones, Caroline P. D.

2011-01-01

Background The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs. Methodology/Principal Findings Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr1175. Phosphorylation of VEGFR2, p38MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo. Conclusions/Significance We conclude that a functional endothelial p38MAPK/Akt/COX-2 signalling axis is required for leptin's pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin's regulation of the vasculature in both non-obese and obese individuals. PMID:21533119

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

PubMed Central

Nemec, Antonia A.; Barchowsky, Aaron

2009-01-01

Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

21 CFR 340.10 - Stimulant active ingredient.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Stimulant active ingredient. 340.10 Section 340.10 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS FOR HUMAN USE STIMULANT DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Active Ingredient § 340.10...

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

PubMed

Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

Angiopoietin-like 4 promotes angiogenesis in the tendon and is increased in cyclically loaded tendon fibroblasts.

PubMed

Mousavizadeh, Rouhollah; Scott, Alex; Lu, Alex; Ardekani, Gholamreza S; Behzad, Hayedeh; Lundgreen, Kirsten; Ghaffari, Mazyar; McCormack, Robert G; Duronio, Vincent

2016-06-01

Angiopoietin-like 4 (ANGPTL4) modulates tendon neovascularization. Cyclic loading stimulates the activity of transforming growth factor-β and hypoxia-inducible factor 1α and thereby increases the expression and release of ANGPTL4 from human tendon cells. Targeting ANGPTL4 and its regulatory pathways is a potential avenue for regulating tendon vascularization to improve tendon healing or adaptation. The mechanisms that regulate angiogenic activity in injured or mechanically loaded tendons are poorly understood. The present study examined the potential role of angiopoietin-like 4 (ANGPTL4) in the angiogenic response of tendons subjected to repetitive mechanical loading or injury. Cyclic stretching of human tendon fibroblasts stimulated the expression and release of ANGPTL4 protein via transforming growth factor-β (TGF-β) and hypoxia-inducible factor 1α (HIF-1α) signalling, and the released ANGPTL4 was pro-angiogenic. Angiogenic activity was increased following ANGPTL4 injection into mouse patellar tendons, whereas the patellar tendons of ANGPTL4 knockout mice displayed reduced angiogenesis following injury. In human rotator cuff tendons, the expression of ANGPTL4 was correlated with the density of tendon endothelial cells. To our knowledge, this is the first study characterizing a role of ANGPTL4 in the tendon. ANGPTL4 may assist in the regulation of vascularity in the injured or mechanically loaded tendon. TGF-β and HIF-1α comprise two signalling pathways that modulate the expression of ANGPTL4 by mechanically stimulated tendon fibroblasts and, in the future, these could be manipulated to influence tendon healing or adaptation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.

PubMed

Koob, Thomas J; Rennert, Robert; Zabek, Nicole; Massee, Michelle; Lim, Jeremy J; Temenoff, Johnna S; Li, William W; Gurtner, Geoffrey

2013-10-01

Human amnion/chorion tissue derived from the placenta is rich in cytokines and growth factors known to promote wound healing; however, preservation of the biological activities of therapeutic allografts during processing remains a challenge. In this study, PURION® (MiMedx, Marietta, GA) processed dehydrated human amnion/chorion tissue allografts (dHACM, EpiFix®, MiMedx) were evaluated for the presence of growth factors, interleukins (ILs) and tissue inhibitors of metalloproteinases (TIMPs). Enzyme-linked immunosorbent assays (ELISA) were performed on samples of dHACM and showed quantifiable levels of the following growth factors: platelet-derived growth factor-AA (PDGF-AA), PDGF-BB, transforming growth factor α (TGFα), TGFβ1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), placental growth factor (PLGF) and granulocyte colony-stimulating factor (GCSF). The ELISA assays also confirmed the presence of IL-4, 6, 8 and 10, and TIMP 1, 2 and 4. Moreover, the relative elution of growth factors into saline from the allograft ranged from 4% to 62%, indicating that there are bound and unbound fractions of these compounds within the allograft. dHACM retained biological activities that cause human dermal fibroblast proliferation and migration of human mesenchymal stem cells (MSCs) in vitro. An in vivo mouse model showed that dHACM when tested in a skin flap model caused mesenchymal progenitor cell recruitment to the site of implantation. The results from both the in vitro and in vivo experiments clearly established that dHACM contains one or more soluble factors capable of stimulating MSC migration and recruitment. In summary, PURION® processed dHACM retains its biological activities related to wound healing, including the potential to positively affect four distinct and pivotal physiological processes intimately involved in wound healing: cell proliferation, inflammation, metalloproteinase activity and recruitment of progenitor cells. This suggests a paracrine mechanism of action for dHACM when used for wound healing applications. ©2013 The Authors. International Wound Journal published by John Wiley & Sons Ltd and Medicalhelplines.com Inc.

TNF-{alpha} promotes cell survival through stimulation of K{sup +} channel and NF{kappa}B activity in corneal epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Ling; Reinach, Peter; Lu, Luo

2005-11-15

Tumor necrosis factor (TNF-{alpha}) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-{alpha} also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-{alpha} stimulation induced activation of a voltage-gated K{sup +} channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-{alpha} on downstream events included NF{kappa}B nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-{alpha} induced increases inmore » p21 expression resulting in partial cell cycle attenuation in the G{sub 1} phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-{alpha}-induced K{sup +} channel activity effectively prevented NF{kappa}B nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-{alpha}. In conclusion, TNF-{alpha} promotes survival of HCE cells through sequential stimulation of K{sup +} channel and NF{kappa}B activities. This response to TNF-{alpha} is dependent on stimulating K{sup +} channel activity because following suppression of K{sup +} channel activity TNF-{alpha} failed to activate NF{kappa}B nuclear translocation and binding to nuclear DNA.« less

Niemann-Pick disease, Type C: evidence for the deficiency of an activating factor stimulating sphingomyelin and glucocerebroside degradation.

PubMed

Christomanou, H

1980-10-01

1) Qualitative lipid analyses by thin-layer chromatography of 4 Niemann-Pick type C spleens confirmed sphingomyelin accumulation together with increase in the amount of glucocerebroside. 2) In the presence of crude sodium taurocholate as detergent, sphingomyelin degradation rates of normal and Niemann-Pick type C-cultured fibroblasts were fairly close under standard conditions at pH 5.0. In the absence of sodium taurocholate, sphingomyelinase activity was optimal at pH 4.0. Sphingomyelinase activities of fibroblasts from two patients with Niemann-Pick disease type C measured without detergent, were about 30% of that of controls. 3) Extracts from Gaucher spleen heated to 90 degrees C and devoid of sphingomyelinase activity stimulated at the optimal pH of 4.0 sphingomyelin degradation by cultured normal fibroblasts (2--4-fold, Niemann-Pick type C fibroblasts (5--9-fold), whereas similarly treated extracts from Niemann-Pick type C spleen showed no stimulation of sphingomyelin catabolism. Heated extracts from normal human spleen exhibited a smaller stimulation than that shown by Gaucher spleen. This stimulating effect could not be observed in fibroblasts from patients suffering from Niemann-Pick type B (sphingomyelinase defect). 4) Heat-treated extracts of Gaucher spleen were fractionated by ion exchange chromatography, isoelectric focusing and gel filtration. The active fractions obtained by these procedures stimulated sphingomyelin as well as glucocerebroside degradation and were absent from the corresponding Niemann-Pick type C preparations. Enriched activator preparations of Gaucher spleen stimulated sphingomyelinase activity of Niemann-Pick type C fibroblasts 25--38-fold and that of normal cells 3-fold. 5) The activating factor had an isoelectric point of 4.0 and an apparent molecular weight, as estimated by gel filtration, of 25000. Treatment with pronase E abolished its activity.

UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells

PubMed Central

Samudio, Ismael; Rezvani, Katayoun; Shaim, Hila; Hofs, Elyse; Ngom, Mor; Bu, Luke; Liu, Guoyu; Lee, Jason T. C.; Imren, Suzan; Lam, Vivian; Poon, Grace F. T.; Ghaedi, Maryam; Takei, Fumio; Humphries, Keith; Jia, William

2016-01-01

Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML. PMID:26941401

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

PubMed

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein

PubMed Central

Ricote, Mercedes; Huang, Jannet; Fajas, Luis; Li, Andrew; Welch, John; Najib, Jamila; Witztum, Joseph L.; Auwerx, Johan; Palinski, Wulf; Glass, Christopher K.

1998-01-01

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ in RAW 264.7 macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis. PMID:9636198

Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells

PubMed Central

2010-01-01

Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory. PMID:20680536

Invasive Cortical Stimulation to Promote Recovery of Function After Stroke

PubMed Central

Plow, Ela B.; Carey, James R.; Nudo, Randolph J.; Pascual-Leone, Alvaro

2011-01-01

Background and Purpose Residual motor deficits frequently linger after stroke. Search for newer effective strategies to promote functional recovery is ongoing. Brain stimulation, as a means of directing adaptive plasticity, is appealing. Animal studies and Phase I and II trials in humans have indicated safety, feasibility, and efficacy of combining rehabilitation and concurrent invasive cortical stimulation. However, a recent Phase III trial showed no advantage of the combination. We critically review results of various trials and discuss the factors that contributed to the distinctive result. Summary of Review Regarding cortical stimulation, it is important to determine the (1) location of peri-infarct representations by integrating multiple neuroanatomical and physiological techniques; (2) role of other mechanisms of stroke recovery; (3) viability of peri-infarct tissue and descending pathways; (4) lesion geometry to ensure no alteration/displacement of current density; and (5) applicability of lessons generated from noninvasive brain stimulation studies in humans. In terms of combining stimulation with rehabilitation, we should understand (1) the principle of homeostatic plasticity; (2) the effect of ongoing cortical activity and phases of learning; and (3) that subject-specific intervention may be necessary. Conclusions Future cortical stimulation trials should consider the factors that may have contributed to the peculiar results of the Phase III trial and address those in future study designs. PMID:19359643

ARL11 regulates lipopolysaccharide-stimulated macrophage activation by promoting mitogen-activated protein kinase (MAPK) signaling.

PubMed

Arya, Subhash B; Kumar, Gaurav; Kaur, Harmeet; Kaur, Amandeep; Tuli, Amit

2018-06-22

A DP- r ibosylation factor- l ike GTPase 11 ( ARL11 ) is a cancer-predisposing gene that has remained functionally uncharacterized to date. In this study, we report that ARL11 is endogenously expressed in mouse and human macrophages and regulates their activation in response to lipopolysaccharide (LPS) stimulation. Accordingly, depletion of ARL11 impaired both LPS-stimulated pro-inflammatory cytokine production by macrophages and their ability to control intracellular replication of Salmonella. LPS-stimulated activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) was substantially compromised in Arl11 -silenced macrophages. In contrast, increased expression of ARL11 led to constitutive ERK1/2 phosphorylation, resulting in macrophage exhaustion. Finally, we found that ARL11 forms a complex with phospho-ERK in macrophages within minutes of LPS stimulation. Taken together, our findings establish ARL11 as a novel regulator of ERK signaling in macrophages, required for macrophage activation and immune function. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitogenic signals and transforming potential of Nyk, a newly identified neural cell adhesion molecule-related receptor tyrosine kinase.

PubMed Central

Ling, L; Kung, H J

1995-01-01

Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family. PMID:8524223

Cell plasticity in wound healing: paracrine factors of M1/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts

PubMed Central

2013-01-01

Background Macrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs). Results HDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production. Conclusions Paracrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair. PMID:23601247

Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp; Takehara, Toshiyuki; Onodera, Yuta

2012-01-13

Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells suchmore » as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.« less

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawake, Shota; Tajima, Noriaki; Mortimer, Jenny C.

Humans are unable to synthesize L-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels,more » leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.« less

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

PubMed Central

Sawake, Shota; Tajima, Noriaki; Lao, Jeemeng; Ishikawa, Toshiki; Yu, Xiaolan; Yamanashi, Yukiko; Yoshimi, Yoshihisa; Kawai-Yamada, Maki

2015-01-01

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity. PMID:26672069

Inhibitory effect of morphine on granulocyte stimulation by tumor necrosis factor and substance P.

PubMed

Stefano, G B; Kushnerik, V; Rodriquez, M; Bilfinger, T V

1994-04-01

We demonstrate that morphine, at higher concentrations than that effective in the inhibition of spontaneously active cells, can antagonize stimulation of human granulocytes by tumor necrosis factor (TNF) or substance P. The antagonistic effect appears to occur indirectly by way of downregulation of the cells' responsiveness to these stimulatory substances. We have previously shown that neutral endopeptidase 24.11 (NEP) is an important enzyme in neuro- and autoimmunoregulation of both vertebrates and invertebrates, and that activation of human granulocytes by monokines and neuropeptides results in regulation of NEP. Exposure of intact human granulocytes to morphine increases NEP by a naloxone-sensitive mechanism. The increased expression of NEP downregulates the stimulatory effect of substance P and TNF. In the case of substance P, we demonstrate the significance of NEP in modulating the process of downregulation by use of a specific NEP inhibitor, phosphoramidon. These results indicate that morphine is a significant factor in downregulating immunocyte responsiveness to NEP substrates and also to those signal molecules (i.e. cytokines) not metabolized by it. In summary, we infer that opiates may be endogenous signal molecules, a status that appears to be amply supported by their immunosuppressive actions.

A novel anti-inflammatory drug, SDZ ASM 981, for the treatment of skin diseases: in vitro pharmacology.

PubMed

Grassberger, M; Baumruker, T; Enz, A; Hiestand, P; Hultsch, T; Kalthoff, F; Schuler, W; Schulz, M; Werner, F J; Winiski, A; Wolff, B; Zenke, G

1999-08-01

SDZ ASM 981, a novel ascomycin macrolactam derivative, has high anti-inflammatory activity in animal models of allergic contact dermatitis and shows clinical efficacy in atopic dermatitis, allergic contact dermatitis and psoriasis, after topical application. Here we report on the in vitro activities of this promising new drug. SDZ ASM 981 inhibits the proliferation of human T cells after antigen-specific or non-specific stimulation. It downregulates the production of Th1 [interleukin (IL)-2, interferon-gamma] and Th2 (IL-4, IL-10) type cytokines after antigen-specific stimulation of a human T-helper cell clone isolated from the skin of an atopic dermatitis patient. SDZ ASM 981 inhibits the phorbol myristate acetate/phytohaemagglutinin-stimulated transcription of a reporter gene coupled to the human IL-2 promoter in the human T-cell line Jurkat and the IgE/antigen-mediated transcription of a reporter gene coupled to the human tumour necrosis factor (TNF)-alpha promoter in the murine mast-cell line CPII. It does not, however, affect the human TNF-alpha promoter controlled transcription of a reporter gene in a murine dendritic cell line (DC18 RGA) after stimulation via the FcgammaRIII receptor. SDZ ASM 981 also prevents the release of preformed pro-inflammatory mediators from mast cells, as shown in the murine cell line CPII after stimulation with IgE/antigen. In summary, these results demonstrate that SDZ ASM 981 is a specific inhibitor of the production of pro-inflammatory cytokines from T cells and mast cells in vitro.

Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.

PubMed Central

Cuenda, A; Alonso, G; Morrice, N; Jones, M; Meier, R; Cohen, P; Nebreda, A R

1996-01-01

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6. Images PMID:8861944

Stimulation of plasmin activity in cultured human fibroblast cells by Porphyromonas endodontalis.

PubMed

Oikawa, T; Ogura, N; Akiba, M; Abiko, Y; Takiguchi, H; Izumi, H

1993-09-01

1. Plasmin activity in the conditioned medium of Gin-1 cells, a human gingival fibroblast cell line, was stimulated by Porphyromonas endodontalis, a putative pathogen of oral submucous abscesses, in a time- and dose-dependent manner. 2. P. endodontalis stimulated the activity of plasminogen activator in both the conditioned medium and the cell lysate. The plasminogen activator in Gin-1 cells was approx. 50 kDa by zymography. 3. The conditioned medium of Gin-1 cells exposed to P. endodontalis stimulated the conversion of human serum prekallikrein to kallikrein. 4. These results suggested that P. endodontalis stimulates the plasminogen activator-plasmin system in Gin-1 cells, and that activated plasmin plays a role in the progress of periodontal tissue inflammation.

White Tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre)-adipocytes

PubMed Central

Söhle, Jörn; Knott, Anja; Holtzmann, Ursula; Siegner, Ralf; Grönniger, Elke; Schepky, Andreas; Gallinat, Stefan; Wenck, Horst; Stäb, Franz; Winnefeld, Marc

2009-01-01

Background The dramatic increase in obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. To investigate how natural substances influence lipolysis and adipogenesis, we determined the effects of White Tea extract on cultured human subcutaneous preadipocytes and adipocytes. Methods For our in vitro studies we used a White Tea extract solution that contained polyphenols and methylxanthines. Utilizing cultured human preadipocytes we investigated White Tea extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. In vitro studies on human adipocytes were performed aiming to elucidate the efficacy of White Tea extract solution to stimulate lipolytic activity. To characterize White Tea extract solution-mediated effects on a molecular level, we analyzed gene expression of essential adipogenesis-related transcription factors by qRT-PCR and determined the expression of the transcription factor ADD1/SREBP-1c on the protein level utilizing immunofluorescence analysis. Results Our data show that incubation of preadipocytes with White Tea extract solution significantly decreased triglyceride incorporation during adipogenesis in a dose-dependent manner (n = 10) without affecting cell viability (n = 10). These effects were, at least in part, mediated by EGCG (n = 10, 50 μM). In addition, White Tea extract solution also stimulated lipolytic activity in adipocytes (n = 7). Differentiating preadipocytes cultivated in the presence of 0.5% White Tea extract solution showed a decrease in PPARγ, ADD1/SREBP-1c, C/EBPα and C/EBPδ mRNA levels. Moreover, the expression of the transcription factor ADD1/SREBP-1c was not only decreased on the mRNA but also on the protein level. Conclusion White Tea extract is a natural source that effectively inhibits adipogenesis and stimulates lipolysis-activity. Therefore, it can be utilized to modulate different levels of the adipocyte life cycle. PMID:19409077

Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis.

PubMed

Oakley, Fiona; Meso, Muriel; Iredale, John P; Green, Karen; Marek, Carylyn J; Zhou, Xiaoying; May, Michael J; Millward-Sadler, Harry; Wright, Matthew C; Mann, Derek A

2005-01-01

Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of kappaB kinase suppressor sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Treatment of hepatic stellate cells with sulfasalazine (0.5-2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle alpha-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-kappaB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45beta, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of kappaB kinase/nuclear factor-kappaB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of kappaB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Inhibition of the inhibitor of kappaB kinase/nuclear factor-kappaB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of kappaB kinase have potential as antifibrotics.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

PubMed

Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

2013-07-01

The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

Shift from posttranscriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human Jurkat cells.

PubMed

Razanajaona, D; Maroc, C; Lopez, M; Mannoni, P; Gabert, J

1992-05-01

The expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is differentially regulated in various cell types. We investigated the mechanisms controlling its expression in 12-O-tetradecanoylphorbol-13-acetate plus phytohemagglutinin-stimulated Jurkat cells, a human T-cell line. In unstimulated cells, GM-CSF mRNA was undetectable by Northern blot. Upon activation, it was detected from 3 h onward, with a progressive increase in the levels of the transcript up to 24 h of stimulation. Whereas cycloheximide treatment at the time of stimulation blocked mRNA induction, its addition at later times resulted in a marked increase in transcript levels. Run-on analysis showed that transcription of the GM-CSF gene was low to undetectable in unstimulated cells; stimulation led to transcriptional activation, which was weak at 6 h but had increased 16-fold at 24 h. In addition, the mRNA half-life decreased during activation, from 2.5 h at 6 h down to 45 min at 24 h. Cycloheximide treatment increased GM-CSF mRNA half-life (3- and 4-fold, respectively). Our results show: (a) both transcriptional and posttranscriptional signals regulate GM-CSF mRNA levels in activated Jurkat cells, (b) de novo protein synthesis is required for mRNA induction, whereas destabilizing labile proteins control the transcript stability, and (c) a shift from a posttranscriptional to a predominant transcriptional control of GM-CSF gene expression occurs during activation.

Proteinase-activated receptor-2 stimulates prostaglandin production in keratinocytes: analysis of prostaglandin receptors on human melanocytes and effects of PGE2 and PGF2alpha on melanocyte dendricity.

PubMed

Scott, Glynis; Leopardi, Sonya; Printup, Stacey; Malhi, Namrita; Seiberg, Miri; Lapoint, Randi

2004-05-01

Prostaglandins (PG) are key mediators of diverse functions in the skin and several reports suggest that PG mediate post-inflammatory pigmentary changes through modulation of melanocyte dendricity and melanin synthesis. The proteinase-activated receptor 2 (PAR-2) is important for skin pigmentation because activation of keratinocyte PAR-2 stimulates uptake of melanosomes through phagocytosis in a Rho-dependent manner. In this report, we show that activation of keratinocyte PAR-2 stimulates release of PGE(2) and PGF(2alpha) and that PGE(2) and PGF(2alpha) act as paracrine factors that stimulate melanocyte dendricity. We characterized the expression of the EP and FP receptors in human melanocytes and show that human melanocytes express EP1 and EP3, and the FP receptor, but not EP2 and EP4. Treatment of melanocytes with EP1 and EP3 receptor agonists resulted in increased melanocyte dendricity, indicating that both EP1 and EP3 receptor signaling contribute to PGE(2)-mediated melanocyte dendricity. Certain EP3 receptor subtypes have been shown to increase adenosine 3',5'-cyclic monophosphate (cAMP) through coupling to Gs, whereas EP1 is known to couple to Gq to activate phospholipase C with elevation in Ca(2+). The cAMP/protein kinase A system is known to modulate melanocyte dendrite formation through modulation of Rac and Rho activity. Neither PGF(2alpha) or PGE(2) elevated cAMP in human melanocytes showing that dendricity observed in response to PGE(2) and PGF(2alpha) is cAMP-independent. Our data suggest that PAR-2 mediates cutaneous pigmentation both through increased uptake of melanosomes by keratinocytes, as well as by release of PGE(2) and PGF(2alpha) that stimulate melanocyte dendricity through EP1, EP3, and FP receptors.

Monocyte Tumor Necrosis Factor-α–Converting Enzyme Catalytic Activity and Substrate Shedding in Sepsis and Noninfectious Systemic Inflammation*

PubMed Central

O’Callaghan, David J. P.; O’Dea, Kieran P.; Scott, Alasdair J.; Takata, Masao

2015-01-01

Objectives: To determine the effect of severe sepsis on monocyte tumor necrosis factor-α–converting enzyme baseline and inducible activity profiles. Design: Observational clinical study. Setting: Mixed surgical/medical teaching hospital ICU. Patients: Sixteen patients with severe sepsis, 15 healthy volunteers, and eight critically ill patients with noninfectious systemic inflammatory response syndrome. Interventions: None. Measurements and Main Results: Monocyte expression of human leukocyte antigen-D-related peptide, sol-tumor necrosis factor production, tumor necrosis factor-α–converting enzyme expression and catalytic activity, tumor necrosis factor receptor 1 and 2 expression, and shedding at 48-hour intervals from day 0 to day 4, as well as p38-mitogen activated protein kinase expression. Compared with healthy volunteers, both sepsis and systemic inflammatory response syndrome patients’ monocytes expressed reduced levels of human leukocyte antigen-D-related peptide and released less sol-tumor necrosis factor on in vitro lipopolysaccharide stimulation, consistent with the term monocyte deactivation. However, patients with sepsis had substantially elevated levels of basal tumor necrosis factor-α–converting enzyme activity that were refractory to lipopolysaccharide stimulation and this was accompanied by similar changes in p38-mitogen activated protein kinase signaling. In patients with systemic inflammatory response syndrome, monocyte basal tumor necrosis factor-α–converting enzyme, and its induction by lipopolysaccharide, appeared similar to healthy controls. Changes in basal tumor necrosis factor-α–converting enzyme activity at day 0 for sepsis patients correlated with Acute Physiology and Chronic Health Evaluation II score and the attenuated tumor necrosis factor-α–converting enzyme response to lipopolysaccharide was associated with increased mortality. Similar changes in monocyte tumor necrosis factor-α–converting enzyme activity could be induced in healthy volunteer monocytes using an in vitro two-hit inflammation model. Patients with sepsis also displayed reduced shedding of monocyte tumor necrosis factor receptors upon stimulation with lipopolysaccharide. Conclusions: Monocyte tumor necrosis factor-α–converting enzyme catalytic activity appeared altered by sepsis and may result in reduced shedding of tumor necrosis factor receptors. Changes seemed specific to sepsis and correlated with illness severity. A better understanding of how tumor necrosis factor-α–converting enzyme function is altered during sepsis will enhance our understanding of sepsis pathophysiology, which will help in the assessment of patient inflammatory status and ultimately may provide new strategies to treat sepsis. PMID:25867908

(+/-)-3-[4-(2-dimethylamino-1-methylethoxy)-phenyl]-1H-pyrazolo[3,4- B]pyridine-1-acetic acid (Y-25510) stimulates production of IL-1 beta and IL-6 at the level of messenger RNA expression in cultured human monocytes.

PubMed

Kusuhara, H; Komatsu, H; Hisadome, M; Ikeda, Y

1996-12-01

(+/-)-3-[4-(2-Dimethylamino-1-methylethoxy)phenyl]-1H-pyrazolo[3, 4-b]pyridine-1-acetic acid (Y-25510) stimulated the mRNA expression for interleukin-1 beta (IL-1 beta), and enhanced the expression induced by lipopolysaccharide (LPS) in cultured human peripheral blood mononuclear cells (PBMC) and THP-1 cells, a cell-line derived from human monocytic leukemia. Y-25510 also stimulated the mRNA expression for IL-6 in both types of the cells, however, the stimulation required the presence of LPS. In THP-1 cells, the stimulation of IL-1 beta mRNA expression by Y-25510 was suppressed by cycloheximide, an inhibitor of protein synthesis. This phenomenon indicates that the stimulation requires de norv protein synthesis. In contrast, the stimulation of mRNA expression for IL-6 by Y-25510 was not suppressed by cycloheximide but suppressed by N alpha-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of nuclear transcription factor-kappa B (NF-kappa B) activation, in the presence of LPS, suggesting that the stimulation requires NF-kappa activation. These results demonstrate that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms. Dexamethasone suppressed the LPS-induced expression of mRNA for IL-1 beta and IL-6 in THP-1 cells, whereas the drug never suppressed the mRNA expression for these cytokines in the presence of Y-25510. The result indicates that Y-25510 stimulates the mRNA expression for IL-1 beta and IL-6 by different mechanisms from those of LPS.

Direct electrical stimulation of human cortex evokes high gamma activity that predicts conscious somatosensory perception

NASA Astrophysics Data System (ADS)

Muller, Leah; Rolston, John D.; Fox, Neal P.; Knowlton, Robert; Rao, Vikram R.; Chang, Edward F.

2018-04-01

Objective. Direct electrical stimulation (DES) is a clinical gold standard for human brain mapping and readily evokes conscious percepts, yet the neurophysiological changes underlying these percepts are not well understood. Approach. To determine the neural correlates of DES, we stimulated the somatosensory cortex of ten human participants at frequency-amplitude combinations that both elicited and failed to elicit conscious percepts, meanwhile recording neural activity directly surrounding the stimulation site. We then compared the neural activity of perceived trials to that of non-perceived trials. Main results. We found that stimulation evokes distributed high gamma activity, which correlates with conscious perception better than stimulation parameters themselves. Significance. Our findings suggest that high gamma activity is a reliable biomarker for perception evoked by both natural and electrical stimuli.

Saturation in Phosphene Size with Increasing Current Levels Delivered to Human Visual Cortex.

PubMed

Bosking, William H; Sun, Ping; Ozker, Muge; Pei, Xiaomei; Foster, Brett L; Beauchamp, Michael S; Yoshor, Daniel

2017-07-26

Electrically stimulating early visual cortex results in a visual percept known as a phosphene. Although phosphenes can be evoked by a wide range of electrode sizes and current amplitudes, they are invariably described as small. To better understand this observation, we electrically stimulated 93 electrodes implanted in the visual cortex of 13 human subjects who reported phosphene size while stimulation current was varied. Phosphene size increased as the stimulation current was initially raised above threshold, but then rapidly reached saturation. Phosphene size also depended on the location of the stimulated site, with size increasing with distance from the foveal representation. We developed a model relating phosphene size to the amount of activated cortex and its location within the retinotopic map. First, a sigmoidal curve was used to predict the amount of activated cortex at a given current. Second, the amount of active cortex was converted to degrees of visual angle by multiplying by the inverse cortical magnification factor for that retinotopic location. This simple model accurately predicted phosphene size for a broad range of stimulation currents and cortical locations. The unexpected saturation in phosphene sizes suggests that the functional architecture of cerebral cortex may impose fundamental restrictions on the spread of artificially evoked activity and this may be an important consideration in the design of cortical prosthetic devices. SIGNIFICANCE STATEMENT Understanding the neural basis for phosphenes, the visual percepts created by electrical stimulation of visual cortex, is fundamental to the development of a visual cortical prosthetic. Our experiments in human subjects implanted with electrodes over visual cortex show that it is the activity of a large population of cells spread out across several millimeters of tissue that supports the perception of a phosphene. In addition, we describe an important feature of the production of phosphenes by electrical stimulation: phosphene size saturates at a relatively low current level. This finding implies that, with current methods, visual prosthetics will have a limited dynamic range available to control the production of spatial forms and that more advanced stimulation methods may be required. Copyright © 2017 the authors 0270-6474/17/377188-10$15.00/0.

Role of the epidermal growth factor receptor in signaling strain-dependent activation of the brain natriuretic peptide gene.

PubMed

Anderson, Hope D I; Wang, Feng; Gardner, David G

2004-03-05

The epidermal growth factor receptor (EGFR) and ectoshedding of heparin-binding epidermal growth factor (HBEGF), an EGFR ligand, have been linked to the development of cardiac myocyte hypertrophy. However, the precise role that the liganded EGFR plays in the transcriptional activation of the gene program that accompanies hypertrophy remains undefined. Utilizing the human (h) BNP gene as a model of hypertrophy-dependent gene activation, we show that activation of the EGFR plays an important role in mediating mechanical strain-dependent stimulation of the hBNP promoter. Strain promotes endothelin (ET) generation through NAD(P)H oxidase-dependent production of reactive oxygen species. ET in turn induces metalloproteinase-mediated cleavage of pro-HBEGF and ectoshedding of HBEGF, which activates the EGFR and stimulates hBNP promoter activity. HBEGF also stimulates other phenotypic markers of hypertrophy including protein synthesis and sarcomeric assembly. The antioxidant N-acetylcysteine or the NAD(P)H oxidase inhibitor, apocynin, inhibited strain-dependent activation of the ET-1 promoter, HBEGF shedding, and hBNP promoter activation. The metalloproteinase inhibitor, GM-6001, prevented the induction of HBEGF ectoshedding and the hBNP promoter response to strain, suggesting a critical role for the metalloproteinase-dependent cleavage event in signaling the strain response. These findings suggest that metalloproteinase activity as an essential step in this pathway may prove to be a relevant therapeutic target in the management of cardiac hypertrophy.

Anti-inflammatory effect of intravenous immunoglobulin in comparison with dexamethasone in vitro: implication for treatment of Kawasaki disease.

PubMed

Makata, Haruyuki; Ichiyama, Takashi; Uchi, Ryutaro; Takekawa, Tsuyoshi; Matsubara, Tomoyo; Furukawa, Susumu

2006-08-01

High-dose intravenous immunoglobulin (IVIG) is a well-established standard therapy for Kawasaki disease (KD) that reduces the risk of developing coronary artery aneurysms. On the other hand, some reports have recommended an alternative therapy with steroids for KD patients. In this study we investigated the anti-inflammatory effect of IVIG in comparison with dexamethasone at clinical doses in vitro. High-dose IVIG inhibited tumor necrosis factor-alpha (TNF-alpha)-induced activation of nuclear factor-kappaB (NF-kappaB) to a greater degree than dexamethasone in human monocytic U937 cells and human coronary arterial endothelial cells (HCAEC), but not in human T lymphocytic Jurkat cells. IVIG was more potent than dexamethasone in reducing the expression of CD16 (FcgammaRIII) in human monocytic THP-1 cells stimulated with lipopolysaccharide and in Jurkat cells stimulated with dimethyl sulfoxide. In HCAEC exposed to TNF-alpha, IVIG and dexamethasone inhibited interleukin-6 production to a similar degree, whereas the expression of E-selectin was inhibited more strongly by IVIG. Our results show that high-dose IVIG inhibits the activation of monocytes/macrophages and coronary arterial endothelial cells more strongly than that of T cells, whereas dexamethasone inhibits the activation of all three cell types. These findings suggest that IVIG or dexamethasone therapy should be chosen to match the types of cells that are activated during acute KD.

Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

PubMed Central

Galasinski, Shelly K.; Lively, Tricia N.; Grebe de Barron, Alexandra; Goodrich, James A.

2000-01-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression. PMID:10688640

Acetyl coenzyme A stimulates RNA polymerase II transcription and promoter binding by transcription factor IID in the absence of histones.

PubMed

Galasinski, S K; Lively, T N; Grebe De Barron, A; Goodrich, J A

2000-03-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression.

Ginkgolide B Reduces LOX-1 Expression by Inhibiting Akt Phosphorylation and Increasing Sirt1 Expression in Oxidized LDL-Stimulated Human Umbilical Vein Endothelial Cells

PubMed Central

Chen, Beidong; Li, Xingguang; Qi, Ruomei

2013-01-01

Oxidized low-density lipoprotein (ox-LDL) is an important risk factor in the development of atherosclerosis. LOX-1, a lectin-like receptor for ox-LDL, is present primarily on endothelial cells and upregulated by ox-LDL, tumor necrosis factor a, shear stress, and cytokines in atherosclerosis. Recent studies demonstrated that ginkgolide B, a platelet-activating factor receptor antagonist, has antiinflammatory and antioxidant effects on endothelial and nerve cells. The present study investigated the effects of ginkgolide B on LOX-1 expression and the possible mechanism of action. Our results showed that ginkgolide B inhibited LOX-1 and intercellular cell adhesion molecule-1 (ICAM-1) expression in ox-LDL-stimulated endothelial cells through a mechanism associated with the attenuation of Akt activation. Similar data were obtained by silencing Akt and LY294002. We also evaluated Sirt1 and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. These molecules play a protective role in endothelial cell injury. The results showed that ginkgolide B increased Sirt1 expression in ox-LDL-treated cells. The inhibitory effects of ginkgolide B on LOX-1 and ICAM-1 expression were reduced in Sirt1 siRNA-transfected cells. Nrf2 expression was increased in ox-LDL-treated cells, and ginkgolide B downregulated Nrf2 expression. These results suggest that ginkgolide B reduces Nrf2 expression by inhibiting LOX-1 expression, consequently reducing oxidative stress injury in ox-LDL-stimulated cells. Altogether, these results indicate that the protective effect of ginkgolide B on endothelial cells may be attributable to a decrease in LOX-1 expression and an increase in Sirt1 expression in ox-LDL-stimulated endothelial cells, the mechanism of which is linked to the inhibition of Akt activation. Ginkgolide B may be a multiple-target drug that exerts protective effects in ox-LDL-treated human umbilical vein endothelial cells. PMID:24069345

Activation of sperm EGFR by light irradiation is mediated by reactive oxygen species.

PubMed

Shahar, Shiran; Hillman, Pnina; Lubart, Rachel; Ickowicz, Debby; Breitbart, Haim

2014-01-01

To acquire fertilization competence, spermatozoa must undergo several biochemical and motility changes in the female reproductive tract, collectively called capacitation. Actin polymerization and the development of hyperactivated motility (HAM) are part of the capacitation process. In a recent study, we showed that irradiation of human sperm with visible light stimulates HAM through a mechanism involving reactive-oxygen-species (ROS), Ca(2+) influx, protein kinases A (PKA), and sarcoma protein kinase (Src). Here, we showed that this effect of light on HAM is mediated by ROS-dependent activation of the epidermal growth factor receptor (EGFR). Interestingly, ROS-mediated HAM even when the EGFR was activated by EGF, the physiological ligand of EGFR. Light irradiation stimulated ROS-dependent actin polymerization, and this effect was abrogated by PBP10, a peptide which activates the actin-severing protein, gelsolin, and causes actin-depolymerization in human sperm. Light-stimulated tyrosine phosphorylation of Src-dependent gelsolin, resulting in enhanced HAM. Thus, light irradiation stimulates HAM through a mechanism involving Src-mediated actin polymerization. Light-stimulated HAM and in vitro-fertilization (IVF) rate in mouse sperm, and these effects were mediated by ROS and EGFR. In conclusion, we show here that irradiation of sperm with visible light, enhances their fertilization capacity via a mechanism requiring ROS, EGFR and HAM. © 2014 The American Society of Photobiology.

The granulocyte-macrophage colony-stimulating factor promoter cis-acting element CLE0 mediates induction signals in T cells and is recognized by factors related to AP1 and NFAT.

PubMed Central

Masuda, E S; Tokumitsu, H; Tsuboi, A; Shlomai, J; Hung, P; Arai, K; Arai, N

1993-01-01

Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene in T cells is activated by the combination of phorbol ester (phorbol myristate acetate) and calcium ionophore (A23187), which mimic antigen stimulation through the T-cell receptor. We have previously shown that a fragment containing bp -95 to +27 of the mouse GM-CSF promoter can confer inducibility to reporter genes in the human Jurkat T-cell line. Here we use an in vitro transcription system to demonstrate that a cis-acting element (positions -54 to -40), referred to as CLE0, is a target for the induction signals. We observed induction with templates containing intact CLE0 but not with templates with deleted or mutated CLE0. We also observed that two distinct signals were required for the stimulation through CLE0, since only extracts from cells treated with both phorbol myristate acetate and A23187 supported optimal induction. Stimulation probably was mediated by CLE0-binding proteins because depletion of these proteins specifically reduced GM-CSF transcription. One of the binding factors possessed biochemical and immunological features identical to those of the transcription factor AP1. Another factor resembled the T-cell-specific factor NFAT. The characteristics of these two factors are consistent with their involvement in GM-CSF induction. The presence of CLE0-like elements in the promoters of interleukin-3 (IL-3), IL-4, IL-5, GM-CSF, and NFAT sites in the IL-2 promoter suggests that the factors we detected, or related factors that recognize these sites, may account for the coordinate induction of these genes during T-cell activation. Images PMID:8246960

Gingival fibroblasts degrade type I collagen films when stimulated with tumor necrosis factor and interleukin 1: evidence that breakdown is mediated by metalloproteinases.

PubMed

Meikle, M C; Atkinson, S J; Ward, R V; Murphy, G; Reynolds, J J

1989-05-01

We previously suggested that periodontal pathogens might mediate connective tissue degradation in periodontal diseases through the ability of antigens from their cell walls to stimulate cytokine production by circulating mononuclear cells. Such cytokines would then induce metalloproteinase (MP) synthesis by resident gingival cells and thus initiate matrix degradation. In the present investigation human gingival fibroblasts (HGFs) were grown on [14C]-labelled type I collagen films and stimulated with either tumor necrosis factor (TNF) or interleukin-1 (IL-1) for 48 h. Collagenolysis occurred in a dose-dependent manner; the optimal dose for human rTNF alpha was 100 ng/ml and for rIL-1 alpha and rIL-1 beta, 1 ng/ml. Collagen degradation was accompanied by increased synthesis and release of the MPs collagenase, gelatinase and stromelysin, and there was a reduction in free TIMP (tissue inhibitor of metalloproteinases): collagenase and stromelysin were detected in both active and latent forms. Cytokine-stimulated collagenolysis was abolished by the addition of exogenous human rTIMP (5 units/ml). We also measured collagenase and TIMP by ELISAs which recognize all forms of collagenase (latent, active or complexed) and TIMP (free or complexed). These showed that while collagenase activity (0.6-1.2 microgram/ml) correlated with lysis, total TIMP levels remained unchanged at approximately 0.2 microgram/ml. These results demonstrate important roles for MPs and TIMP in regulating type I collagen degradation by HGFs, and support the hypothesis that connective tissue destruction during inflammatory diseases may be initiated, at least in part, by TNF and IL-1.

Regulation of monocarboxylate transporter 1 (MCT1) promoter by butyrate in human intestinal epithelial cells: involvement of NF-kappaB pathway.

PubMed

Borthakur, Alip; Saksena, Seema; Gill, Ravinder K; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

2008-04-01

Butyrate, a short chain fatty acid (SCFA) produced by bacterial fermentation of undigested carbohydrates in the colon, constitutes the major fuel for colonocytes. We have earlier shown the role of apically localized monocarboxylate transporter isoform 1 (MCT1) in transport of butyrate into human colonic Caco-2 cells. In an effort to study the regulation of MCT1 gene, we and others have cloned the promoter region of the MCT1 gene and identified cis elements for key transcription factors. A previous study has shown up-regulation of MCT1 expression, and activity by butyrate in AA/C1 human colonic epithelial cells, however, the detailed mechanisms of this up-regulation are not known. In this study, we demonstrate that butyrate, a substrate for MCT1, stimulates MCT1 promoter activity in Caco-2 cells. This effect was dose dependent and specific to butyrate as other predominant SCFAs, acetate, and propionate, were ineffective. Utilizing progressive deletion constructs of the MCT1 promoter, we showed that the putative butyrate responsive elements are in the -229/+91 region of the promoter. Butyrate stimulation of the MCT1 promoter was found to be independent of PKC, PKA, and tyrosine kinases. However, specific inhibitors of the NF-kappaB pathway, lactacystein (LC), and caffeic acid phenyl ester (CAPE) significantly reduced the MCT1 promoter stimulation by butyrate. Also, butyrate directly stimulated NF-kappaB-dependent luciferase reporter activity. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) also stimulated MCT1 promoter activity, however, unlike butyrate, this stimulation was unaltered by the NF-kappaB inhibitors. Further, the combined effect of butyrate, and TSA on MCT1 promoter activity was additive, indicating that their mechanisms of action were independent. Our results demonstrate the involvement of NF-kappaB pathway in the regulation of MCT1 promoter activity by butyrate. 2007 Wiley-Liss, Inc.

Amino acids exhibit anti-inflammatory effects in human monocytic leukemia cell line, THP-1 cells.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Sonaka, Ichiro; Ohsaki, Ayami; Hirano, Reiji; Haneda, Yasuhiro; Fukano, Reiji; Hara, Masami; Furukawa, Susumu

2011-11-01

The elemental diet is one of the effective therapies for inflammatory bowel disease. However, the mechanism remains unclear, and there have never been reports about the inhibitory effects of amino acids in human monocytes/macrophages. We investigated the inhibitory effects of amino acids on cytokine production or expression of adhesion molecules that are involved in inflammatory diseases, in human monocytes/macrophages. We examined the inhibitory effects of cysteine, histidine or glycine on the induction of nuclear factor-κB (NF-κB) activation, expression of intracellular adhesion molecule-1 (ICAM-1, CD54) and production of interleukin-8 (IL-8) in THP-1 cells, a human monocytic leukemia cell line, and peripheral blood mononuclear cells (PBMCs) stimulated with tumor necrosis factor-α (TNF-α). Cysteine, histidine and glycine significantly reduced the activation of NF-κB in THP-1 cells stimulated with TNF-α. In addition, cysteine and histidine significantly inhibited the expression of ICAM-1 and production of IL-8 in THP-1 cells and PBMCs. Our results suggest that cysteine and histidine exhibit anti-inflammatory effects in THP-1 cells, and may be responsible for the efficacy of treatment in inflammatory bowel diseases.

Notch signaling mediates granulocyte-macrophage colony-stimulating factor priming-induced transendothelial migration of human eosinophils.

PubMed

Liu, L Y; Wang, H; Xenakis, J J; Spencer, L A

2015-07-01

Priming with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances eosinophil migration and exacerbates the excessive accumulation of eosinophils within the bronchial mucosa of asthmatics. However, mechanisms that drive GM-CSF priming are incompletely understood. Notch signaling is an evolutionarily conserved pathway that regulates cellular processes, including migration, by integrating exogenous and cell-intrinsic cues. This study investigates the hypothesis that the priming-induced enhanced migration of human eosinophils requires the Notch signaling pathway. Using pan Notch inhibitors and newly developed human antibodies that specifically neutralize Notch receptor 1 activation, we investigated a role for Notch signaling in GM-CSF-primed transmigration of human blood eosinophils in vitro and in the airway accumulation of mouse eosinophils in vivo. Notch receptor 1 was constitutively active in freshly isolated human blood eosinophils, and inhibition of Notch signaling or specific blockade of Notch receptor 1 activation during GM-CSF priming impaired priming-enhanced eosinophil transendothelial migration in vitro. Inclusion of Notch signaling inhibitors during priming was associated with diminished ERK phosphorylation, and ERK-MAPK activation was required for GM-CSF priming-induced transmigration. In vivo in mice, eosinophil accumulation within allergic airways was impaired following systemic treatment with Notch inhibitor, or adoptive transfer of eosinophils treated ex vivo with Notch inhibitor. These data identify Notch signaling as an intrinsic pathway central to GM-CSF priming-induced eosinophil tissue migration. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

PubMed

Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

2015-01-01

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

Posterior reversible encephalopathy syndrome (PRES) after granulocyte-colony stimulating factor (G-CSF) therapy: a report of 2 cases.

PubMed

Stübgen, Joerg-Patrick

2012-10-15

Two patients with recurrent lymphoma developed an acute, transient encephalopathy following administration of recombinant human granulocyte-colony stimulating factor (rhG-CSF), filgrastim, in anticipation of leukapheresis for hematopoietic stem cell transplantation. Head magnetic resonance imaging showed evidence of blood-brain barrier (BBB) breakdown, compatible with posterior reversible encephalopathy syndrome (PRES). The proposed pathogenesis of PRES was rhG-CSF-induced neutrophil mobilization and activation with the release of inflammatory mediators, resulting in transient alteration of barrier permeability and capillary leakage. Copyright © 2012 Elsevier B.V. All rights reserved.

Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ryu, Jae-Sung; Jung, Yeon-Hwa; Cho, Mi-Young

Highlights: • Co-culture of hSDMSCs with SNP-stimulated chondrocytes improves anti-inflammation. • Co-culture system produces IGF-1. • Co-culture system suppresses inflammatory genes expression. • Co-culture system improves cell proliferation. • Exogenous IGF-1 inhibits inflammatory activity in SNP-stimulated chondrocytes. - Abstract: Rheumatoid arthritis (RA) and osteoarthritis (OA) are primarily chronic inflammatory diseases. Mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the mesodermal lineage, and to regulate immunomodulatory activity. Specifically, MSCs have been shown to secrete insulin-like growth factor 1 (IGF-1). The purpose of the present study was to examine the inhibitory effects on inflammatory activity from a co-culturemore » of human synovium-derived mesenchymal stem cells (hSDMSCs) and sodium nitroprusside (SNP)-stimulated chondrocytes. First, chondrocytes were treated with SNP to generate an in vitro model of RA or OA. Next, the co-culture of hSDMSCs with SNP-stimulated chondrocytes reduced inflammatory cytokine secretion, inhibited expression of inflammation activity-related genes, generated IGF-1 secretion, and increased the chondrocyte proliferation rate. To evaluate the effect of IGF-1 on inhibition of inflammation, chondrocytes pre-treated with IGF-1 were treated with SNP, and then the production of inflammatory cytokines was analyzed. Treatment with IGF-1 was shown to significantly reduce inflammatory cytokine secretion in SNP-stimulated chondrocytes. Our results suggest that hSDMSCs offer a new strategy to promote cell-based cartilage regeneration in RA or OA.« less

Ultra-low power laser stimulation impairs the adhesion of Staphylococcus aureus to primary human cells, and interferes with the expression of staphylococcal pathogenic factors.

PubMed

Petruzzelli, Sabina; Congiu, Antonio; Gallamini, Michele; Pompei, Raffaello

2014-04-01

Lasers are commonly used in several fields of medicine as a complementary therapy for internal medicine, surgery and also diagnostics. The efficacy of ultra-low level laser therapy (ULLLT) at power levels around 0.15 mW/cm(2) has been demonstrated both in in vitro experiments and in the clinical environment. This work used an ULLLT laser source to analyze its efficacy on Staphylococcus aureus adhesion to cells and on its ability to produce pathogenic factors. Laser stimulation succeeded in impairing the binding of S. aureus to primary human cells in culture and in inhibiting the expression of coagulase, one of the main staphylococcal pathogenic factors. The importance of the extracellular matrix (ECM) and the modification of the ECM redox potential in these activities were also evidenced.

Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice

PubMed Central

Alahmari, A. A.; Sreekumar, B.; Patel, V.; Ashat, M.; Alexandre, M.; Uduman, A. K.; Akinbiyi, E. O.; Ceplenski, A.; Shugrue, C. A.; Kolodecik, T. R.; Messenger, S. W.; Groblewski, G. E.; Gorelick, F. S.

2018-01-01

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease. PMID:29870540

Protective effect of nuclear factor E2-related factor 2 on inflammatory cytokine response to brominated diphenyl ether-47 in the HTR-8/SVneo human first trimester extravillous trophoblast cell line.

PubMed

Park, Hae-Ryung; Loch-Caruso, Rita

2014-11-15

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants, and BDE-47 is a prevalent PBDE congener detected in human tissues. Exposure to PBDEs has been linked to adverse pregnancy outcomes in humans. Although the underlying mechanisms of adverse birth outcomes are poorly understood, critical roles for oxidative stress and inflammation are implicated. The present study investigated antioxidant responses in a human extravillous trophoblast cell line, HTR-8/SVneo, and examined the role of nuclear factor E2-related factor 2 (Nrf2), an antioxidative transcription factor, in BDE-47-induced inflammatory responses in the cells. Treatment of HTR-8/SVneo cells with 5, 10, 15, and 20μM BDE-47 for 24h increased intracellular glutathione (GSH) levels compared to solvent control. Treatment of HTR-8/SVneo cells with 20μM BDE-47 for 24h induced the antioxidant response element (ARE) activity, indicating Nrf2 transactivation by BDE-47 treatment, and resulted in differential expression of redox-sensitive genes compared to solvent control. Pretreatment with tert-butyl hydroquinone (tBHQ) or sulforaphane, known Nrf2 inducers, reduced BDE-47-stimulated IL-6 release with increased ARE reporter activity, reduced nuclear factor kappa B (NF-κB) reporter activity, increased GSH production, and stimulated expression of antioxidant genes compared to non-Nrf2 inducer pretreated groups, suggesting that Nrf2 may play a protective role against BDE-47-mediated inflammatory responses in HTR-8/SVneo cells. These results suggest that Nrf2 activation significantly attenuated BDE-47-induced IL-6 release by augmentation of cellular antioxidative system via upregulation of Nrf2 signaling pathways, and that Nrf2 induction may be a potential therapeutic target to reduce adverse pregnancy outcomes associated with toxicant-induced oxidative stress and inflammation. Copyright © 2014 Elsevier Inc. All rights reserved.

Sphingosine 1-phosphate (S1P) suppresses the collagen-induced activation of human platelets via S1P4 receptor.

PubMed

Onuma, Takashi; Tanabe, Kumiko; Kito, Yuko; Tsujimoto, Masanori; Uematsu, Kodai; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Doi, Tomoaki; Nagase, Kiyoshi; Akamatsu, Shigeru; Tokuda, Haruhiko; Ogura, Shinji; Iwama, Toru; Kozawa, Osamu; Iida, Hiroki

2017-08-01

Sphingosine 1-phosphate (S1P) is as an extracellular factor that acts as a potent lipid mediator by binding to specific receptors, S1P receptors (S1PRs). However, the precise role of S1P in human platelets that express S1PRs has not yet been fully clarified. We previously reported that heat shock protein 27 (HSP27) is released from human platelets accompanied by its phosphorylation stimulated by collagen. In the present study, we investigated the effect of S1P on the collagen-induced platelet activation. S1P pretreatment markedly attenuated the collagen-induced aggregation. Co-stimulation with S1P and collagen suppressed collagen-induced platelet activation, but the effect was weaker than that of S1P-pretreatment. The collagen-stimulated secretion of platelet-derived growth factor (PDGF)-AB and the soluble CD40 ligand (sCD40L) release were significantly reduced by S1P. In addition, S1P suppressed the collagen-induced release of HSP27 as well as the phosphorylation of HSP27. S1P significantly suppressed the collagen-induced phosphorylation of p38 mitogen-activated protein kinase. S1P increased the levels of GTP-bound Gαi and GTP-bound Gα13 coupled to S1PPR1 and/or S1PR4. CYM50260, a selective S1PR4 agonist, but not SEW2871, a selective S1PR1 agonist, suppressed the collagen-stimulated platelet aggregation, PDGF-AB secretion and sCD40L release. In addition, CYM50260 reduced the release of phosphorylated-HSP27 by collagen as well as the phosphorylation of HSP27. The selective S1PR4 antagonist CYM50358, which failed to affect collagen-induced HSP27 phosphorylation, reversed the S1P-induced attenuation of HSP27 phosphorylation by collagen. These results strongly suggest that S1P inhibits the collagen-induced human platelet activation through S1PR4 but not S1PR1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sphingosine kinase inhibitor suppresses IL-18-induced interferon-gamma production through inhibition of p38 MAPK activation in human NK cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheon, Soyoung; Song, Seok Bean; Jung, Minkyung

2008-09-12

Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-{gamma} inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-{gamma} production, we measured IL-18-induced IFN-{gamma} production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-{gamma} expression was blocked by SKI pre-treatment in both mRNAmore » and protein levels. In addition, the increased IFN-{gamma} production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-{gamma} production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-{gamma} production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-{gamma} production via p38 MAPK.« less

Differential responsiveness of luteinized human granulosa cells to gonadotropins and insulin-like growth factor I for induction of aromatase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Christman, G.M.; Randolph, J.F. Jr.; Peegel, H.

1991-06-01

The objective of this study was to examine the in vitro responsiveness of cultured luteinized human granulosa cells over time to insulin-like growth factor 1 (IGF-1), human follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) for the induction of aromatase activity. Granulosa cells were retrieved from preovulatory follicles in patients undergoing in vitro fertilization. Cells were cultured for a period of 72 hours or 10 days. The ability of hCG, human FSH, and/or IGF-I to induce aromatase activity was assayed by the stereospecific release of tritium from (1B-3H)androstenedione. Short-term cultures (72 hours) demonstrated a marked rise in aromatase activity inmore » response to human FSH and IGF-I, whereas a smaller response to hCG was observed. In contrast, 10-day cultures demonstrated responsiveness predominantly to hCG rather than human FSH for the induction of aromatase activity with no remarkable effect of IGF-I. Luteinized human granulosa cells undergo a transformation from an initial human FSH and IGF-I responsive state to an hCG responsive state in long-term cultures.« less

Protein concentration in pre-ovulatory follicular fluid related to ovarian stimulation.

PubMed

Suchanek, E; Mujkic-Klaric, A; Grizelj, V; Simunic, V; Kopjar, B

1990-05-01

Sixty follicular fluids obtained from 26 women with either clomiphene citrate and human menopausal gonadotropins (hMG) or hMG-induced ovulation were analyzed for the contents of total proteins, fibrinogen, plasminogen, antithrombin III, ceruloplasmin, alpha-2 macroglobulin, alpha-1 antitrypsin and immunoglobulins (IgG, IgA, IgM). Concentrations of these proteins was correlated to the type of ovarian follicle growth induction. Follicular fluids from patients stimulated with clomiphene citrate-hMG contained significantly higher concentrations of ceruloplasmin than those treated with hMG alone. No significant differences in the concentrations of other proteins were noted between the two types of ovarian induction. A multivariate data analysis resulted in three Varimax factors (VRX I) suggesting that proteins with antiprotease activity in the follicular fluid may play a role in human follicle maturation. Follicular fluid Ig may reflect the degree of follicular wall permeability under hMG treatment. Accordingly, it may be assumed that a combination of different proteins described by VRX factors could be used for evaluation of ovarian stimulation.

The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sekiya, M.; Frohlich, E.D.; Cole, F.E.

1991-01-01

In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation.more » In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.« less

Anti-photoaging effect of aaptamine in UVB-irradiated human dermal fibroblasts and epidermal keratinocytes.

PubMed

Kim, Min-Ji; Woo, Seon Wook; Kim, Myung-Suk; Park, Ji-Eun; Hwang, Jae-Kwan

2014-12-01

Chronic exposure to ultraviolet (UV) irradiation causes sunburn, inflammatory responses, skin cancer, and photoaging. Photoaging, in particular, generates reactive oxygen species (ROS) that stimulate mitogen-activated protein kinase (MAPK) signaling and transcription factors. UV irradiation also activates matrix metalloproteinases (MMPs) expression and inactivates collagen synthesis. Aaptamine, a marine alkaloid isolated from the marine sponge, has been reported to have antitumor, antimicrobial, antiviral, and antioxidant activities. However, the photo-protective effects of aaptamine have not been elucidated. In this study, our data demonstrated that aaptamine deactivated UVB-induced MAPK and activator protein-1 signaling by suppressing ROS, resulting in attenuating the expression of MMPs in UVB-irradiated human dermal fibroblasts. Aaptamine also decreased proinflammatory cytokines such as cyclooxygenase-2, tumor necrosis factor-α, interleukin-1β, and nuclear factor-kappa B subunits in UVB-irradiated human keratinocytes. In conclusion, we suggest that aaptamine represents a novel and effective strategy for treatment and prevention of photoaging.

Infrared neural stimulation of human spinal nerve roots in vivo.

PubMed

Cayce, Jonathan M; Wells, Jonathon D; Malphrus, Jonathan D; Kao, Chris; Thomsen, Sharon; Tulipan, Noel B; Konrad, Peter E; Jansen, E Duco; Mahadevan-Jansen, Anita

2015-01-01

Infrared neural stimulation (INS) is a neurostimulation modality that uses pulsed infrared light to evoke artifact-free, spatially precise neural activity with a noncontact interface; however, the technique has not been demonstrated in humans. The objective of this study is to demonstrate the safety and efficacy of INS in humans in vivo. The feasibility of INS in humans was assessed in patients ([Formula: see text]) undergoing selective dorsal root rhizotomy, where hyperactive dorsal roots, identified for transection, were stimulated in vivo with INS on two to three sites per nerve with electromyogram recordings acquired throughout the stimulation. The stimulated dorsal root was removed and histology was performed to determine thermal damage thresholds of INS. Threshold activation of human dorsal rootlets occurred in 63% of nerves for radiant exposures between 0.53 and [Formula: see text]. In all cases, only one or two monitored muscle groups were activated from INS stimulation of a hyperactive spinal root identified by electrical stimulation. Thermal damage was first noted at [Formula: see text] and a [Formula: see text] safety ratio was identified. These findings demonstrate the success of INS as a fresh approach for activating human nerves in vivo and providing the necessary safety data needed to pursue clinically driven therapeutic and diagnostic applications of INS in humans.

Alternative pathway regulation by factor H modulates Streptococcus pneumoniae induced proinflammatory cytokine responses by decreasing C5a receptor crosstalk.

PubMed

van der Maten, Erika; de Bont, Cynthia M; de Groot, Ronald; de Jonge, Marien I; Langereis, Jeroen D; van der Flier, Michiel

2016-12-01

Bacterial pathogens not only stimulate innate immune receptors, but also activate the complement system. Crosstalk between complement C5a receptor (C5aR) and other innate immune receptors is known to enhance the proinflammatory cytokine response. An important determinant of the magnitude of complement activation is the activity of the alternative pathway, which serves as an amplification mechanism for complement activation. Both alternative pathway activity as well as plasma levels of factor H, a key inhibitor of the alternative pathway, show large variation within the human population. Here, we studied the effect of factor H-mediated regulation of the alternative pathway on bacterial-induced proinflammatory cytokine responses. We used the human pathogen Streptococcus pneumoniae as a model stimulus to induce proinflammatory cytokine responses in human peripheral blood mononuclear cells. Serum containing active complement enhanced pneumococcal induced proinflammatory cytokine production through C5a release and C5aR crosstalk. We found that inhibition of the alternative pathway by factor H, with a concentration equivalent to a high physiological level, strongly reduced C5a levels and decreased proinflammatory cytokine production in human peripheral blood mononuclear cells. This suggests that variation in alternative pathway activity due to variation in factor H plasma levels affects individual cytokine responses during infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis.

PubMed

Sawake, Shota; Tajima, Noriaki; Mortimer, Jenny C; Lao, Jeemeng; Ishikawa, Toshiki; Yu, Xiaolan; Yamanashi, Yukiko; Yoshimi, Yoshihisa; Kawai-Yamada, Maki; Dupree, Paul; Tsumuraya, Yoichi; Kotake, Toshihisa

2015-12-01

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity. © 2015 American Society of Plant Biologists. All rights reserved.

IL-29 Enhances CXCL10 Production in TNF-α-stimulated Human Oral Epithelial Cells.

PubMed

Hosokawa, Yoshitaka; Hosokawa, Ikuko; Shindo, Satoru; Ozaki, Kazumi; Matsuo, Takashi

2017-08-01

Interleukin-29 (IL-29) is a cytokine belonging to the Type III interferon family. It was recently detected in the gingival crevicular fluid of periodontitis patients. However, the role of IL-29 in the pathogenesis of periodontal disease remains unknown. The aim of this study was to examine the effects of IL-29 on C-X-C motif chemokine ligand 10 (CXCL10) production in human oral epithelial cells. We measured CXCL10 production in TR146 cells, which is a human oral epithelial cell line, using an enzyme-linked immunosorbent assay. We used a Western blot analysis to detect IL-29 receptor expression and the phosphorylation levels of signal transduction molecules, including p38 mitogen-activated protein kinases (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor (NF)- κB p65, in the TR146 cells. The TR146 cells expressed the IL-29 receptor. IL-29 induced CXCL10 production in the TR146 cells. IL-29 significantly enhanced CXCL10 production in tumor necrosis factor (TNF)-α-stimulated TR146 cells. The p38 MAPK, STAT3, and NF-κB pathways were found to be related to the IL-29-induced enhancement of CXCL10 production in TNF-α-stimulated TR146 cells. IL-29 promotes T helper 1-cell accumulation in periodontal lesions by inducing CXCL10 production in oral epithelial cells.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

PubMed

Hu, Yae; Yan, Ruhong; Zhang, Ce; Zhou, Zhichao; Liu, Meng; Wang, Can; Zhang, Hong; Dong, Liang; Zhou, Tiantian; Wu, Yi; Dong, Ningzheng; Wu, Qingyu

2018-04-12

Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia. © 2018 American Heart Association, Inc.

HMGB1-stimulated human primary cardiac fibroblasts exert a paracrine action on human and murine cardiac stem cells.

PubMed

Rossini, Alessandra; Zacheo, Antonella; Mocini, David; Totta, Pierangela; Facchiano, Antonio; Castoldi, Raffaella; Sordini, Paolo; Pompilio, Giulio; Abeni, Damiano; Capogrossi, Maurizio C; Germani, Antonia

2008-04-01

High Mobility Box 1 Protein (HMGB1) is a cytokine released into the extracellular space by necrotic cells and activated macrophages in response to injury. We recently demonstrated that HMGB1 administration into the mouse heart during acute myocardial infarction induces cardiac tissue regeneration by activating resident cardiac c-kit+ cells (CSCs) and significantly enhances left ventricular function. In the present study it was analyzed the hypothesis that human cardiac fibroblasts (cFbs) exposed to HMGB1 may exert a paracrine effect on mouse and human CSCs. Human cFbs expressed the HMGB1 receptor RAGE. Luminex technology and ELISA assays revealed that HMGB1 significantly enhanced VEGF, PlGF, Mip-1alpha, IFN-gamma, GM-CSF, Il-10, Il-1beta, Il-4, Il-1ra, Il-9 and TNF-alpha in cFbs cell culture medium. HMGB1-stimulated cFbs conditioned media induced CSC migration and proliferation. These effects were significantly higher to those obtained when HMGB1 was added directly to the culture medium. In conclusion, we provide evidence that HMGB1 may act in a paracrine manner stimulating growth factor, cytokine and chemokine release by cFbs which, in turn, modulate CSC function. Via this mechanism HMGB1 may contribute to cardiac tissue regeneration.

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Briolay, A.; Lencel, P.; Bessueille, L.

2013-01-18

Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification.more » In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.« less

Functional magnetic resonance imaging of the human spinal cord during vibration stimulation of different dermatomes.

PubMed

Lawrence, Jane M; Stroman, Patrick W; Kollias, Spyros S

2008-03-01

We investigated noninvasively areas of the healthy human spinal cord that become active in response to vibration stimulation of different dermatomes using functional magnetic resonance imaging (fMRI). The objectives of this study were to: (1) examine the patterns of consistent activity in the spinal cord during vibration stimulation of the skin, and (2) investigate the rostrocaudal distribution of active pixels when stimulation was applied to different dermatomes. FMRI of the cervical and lumbar spinal cord of seven healthy human subjects was carried out during vibration stimulation of six different dermatomes. In separate experiments, vibratory stimulation (about 50 Hz) was applied to the right biceps, wrist, palm, patella, Achilles tendon and left palm. The segmental distribution of activity observed by fMRI corresponded well with known spinal cord neuroanatomy. The peak number of active pixels was observed at the expected level of the spinal cord with some activity in the adjacent segments. The rostrocaudal distribution of activity was observed to correspond to the dermatome being stimulated. Cross-sectional localization of activity was primarily in dorsal areas but also spread into ventral and intermediate areas of the gray matter and a distinct laterality ipsilateral to the stimulated limb was not observed. We demonstrated that fMRI can detect a dermatome-dependent pattern of spinal cord activity during vibratory stimulation and can be used as a passive stimulus for the noninvasive assessment of the functional integrity of the human spinal cord. Demonstration of cross-sectional selectivity of the activation awaits further methodological and experimental refinements.

Glucocorticoids suppress tumor necrosis factor-alpha expression by human monocytic THP-1 cells by suppressing transactivation through adjacent NF-kappa B and c-Jun-activating transcription factor-2 binding sites in the promoter.

PubMed

Steer, J H; Kroeger, K M; Abraham, L J; Joyce, D A

2000-06-16

Glucocorticoid drugs suppress tumor necrosis factor-alpha (TNF-alpha) synthesis by activated monocyte/macrophages, contributing to an anti-inflammatory action in vivo. In lipopolysaccharide (LPS)-activated human monocytic THP-1 cells, glucocorticoids acted primarily on the TNF-alpha promoter to suppress a burst of transcriptional activity that occurred between 90 min and 3 h after LPS exposure. LPS increased nuclear c-Jun/ATF-2, NF-kappaB(1)/Rel-A, and Rel-A/C-Rel transcription factor complexes, which bound specifically to oligonucleotide sequences from the -106 to -88 base pair (bp) region of the promoter. The glucocorticoid, dexamethasone, suppressed nuclear binding activity of these complexes prior to and during the critical phase of TNF-alpha transcription. Site-directed mutagenesis in TNF-alpha promoter-luciferase reporter constructs showed that the adjacent c-Jun/ATF-2 (-106 to -99 bp) and NF-kappaB (-97 to -88 bp) binding sites each contributed to the LPS-stimulated expression. Mutating both sites largely prevented dexamethasone from suppressing TNF-alpha promoter-luciferase reporters. LPS exposure also increased nuclear Egr-1 and PU.1 abundance. The Egr-1/Sp1 (-172 to -161 bp) binding sites and the PU.1-binding Ets site (-116 to -110 bp) each contributed to the LPS-stimulated expression but not to glucocorticoid response. Dexamethasone suppressed the abundance of the c-Fos/c-Jun complex in THP-1 cell nuclei, but there was no direct evidence for c-Fos/c-Jun transactivation through sites in the -172 to -52 bp region. Small contributions to glucocorticoid response were attributable to promoter sequences outside the -172 to -88 bp region and to sequences in the TNF-alpha 3'-untranslated region. We conclude that glucocorticoids suppress LPS-stimulated secretion of TNF-alpha from human monocytic cells largely through antagonizing transactivation by c-Jun/ATF-2 and NF-kappaB complexes at binding sites in the -106 to -88 bp region of the TNF-alpha promoter.

Klotho modulates FGF23-mediated NO synthesis and oxidative stress in human coronary artery endothelial cells.

PubMed

Richter, Beatrice; Haller, Jacqueline; Haffner, Dieter; Leifheit-Nestler, Maren

2016-09-01

Chronic kidney disease (CKD) is a state of Klotho deficiency and excess of the phosphaturic hormone fibroblast growth factor 23 (FGF23). Both dysregulations were shown to be associated with endothelial dysfunction in humans, but direct vascular effects of FGF23 remain largely elusive. In vitro experiments were performed to assess the effects of FGF23 (10 ng/mL) in relation to its co-receptor Klotho on nitric oxide (NO) synthesis and reactive oxygen species (ROS) formation and detoxification in human coronary artery endothelial cells (HCAEC). Membrane-bound Klotho is expressed in HCAEC, and FGF23 increases the expression of the Klotho shedding protease ADAM17, and consequently the secretion of soluble Klotho. FGF23 activates FGF receptor 1 and stimulates NO release via Akt-dependent activation of endothelial NO synthase (eNOS). Both FGF receptor (FGFR)-dependent ROS formation via activation of NADPH oxidase 2 (Nox2) as well as ROS degradation via superoxide dismutase 2 (SOD2) and catalase (CAT) is stimulated by FGF23. Pre-incubation with a Klotho inhibitor blunts the FGF23-stimulated Akt-eNOS activation and NO synthesis, and decreases ROS degradation by blocking SOD2 and CAT enzymes, whereas FGF23-stimulated ROS synthesis via Nox2 is unaffected, resulting in low NO bioavailability and increased oxidative stress. Our data indicate that in the presence of Klotho, FGF23 induces NO release in HCAEC and its stimulating effects on ROS production are counterbalanced by increased ROS degradation. In states of Klotho deficiency, e.g., CKD, FGF23-mediated NO synthesis is blunted and ROS formation overrules ROS degradation. Thus, FGF23 excess may primarily promote oxidative stress and thus endothelial dysfunction.

Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth Factor Receptors in Human Melanoma Cells

PubMed Central

Fiori, Jennifer L.; Zhu, Tie-Nian; O'Connell, Michael P.; Hoek, Keith S.; Indig, Fred E.; Frank, Brittany P.; Morris, Christa; Kole, Sutapa; Hasskamp, Joanne; Elias, George; Weeraratna, Ashani T.; Bernier, Michel

2009-01-01

The actin-binding protein filamin A (FLNa) affects the intracellular trafficking of various classes of receptors and has a potential role in oncogenesis. However, it is unclear whether FLNa regulates the signaling capacity and/or down-regulation of the activated epidermal growth factor receptor (EGFR). Here it is shown that partial knockdown of FLNa gene expression blocked ligand-induced EGFR responses in metastatic human melanomas. To gain greater insights into the role of FLNa in EGFR activation and intracellular sorting, we used M2 melanoma cells that lack endogenous FLNa and a subclone in which human FLNa cDNA has been stably reintroduced (M2A7 cells). Both tyrosine phosphorylation and ubiquitination of EGFR were significantly lower in epidermal growth factor (EGF)-stimulated M2 cells when compared with M2A7 cells. Moreover, the lack of FLNa interfered with EGFR interaction with the ubiquitin ligase c-Cbl. M2 cells exhibited marked resistance to EGF-induced receptor degradation, which was very active in M2A7 cells. Despite comparable rates of EGF-mediated receptor endocytosis, internalized EGFR colocalized with the lysosomal marker lysosome-associated membrane protein-1 in M2A7 cells but not M2 cells, in which EGFR was found to be sequestered in large vesicles and subsequently accumulated in punctated perinuclear structures after EGF stimulation. These results suggest the requirement of FLNa for efficient EGFR kinase activation and the sorting of endocytosed receptors into the degradation pathway. PMID:19213840

Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khaiboullina, Svetlana F., E-mail: sv.khaiboullina@gmail.com; Morzunov, Sergey P.; Boichuk, Sergei V.

2013-09-01

Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirusmore » triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.« less

Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

NASA Astrophysics Data System (ADS)

Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

2005-05-01

Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

Tacrolimus potently inhibits human osteoclastogenesis induced by IL-17 from human monocytes alone and suppresses human Th17 differentiation.

PubMed

Yago, Toru; Nanke, Yuki; Kawamoto, Manabu; Yamanaka, Hisashi; Kotake, Shigeru

2012-08-01

Tacrolimus (FK506, Prograf®) is an orally available, T cell specific and anti-inflammatory agent that has been proposed as a therapeutic drug in rheumatoid arthritis (RA) patients. It has been known that T cells have a critical role in the pathogenesis of RA. Recent studies suggest that Th17 cells, which mainly produce IL-17, are involved in many autoimmune inflammatory disease including RA. The present study was undertaken to assess the effect of tacrolimus on IL-17-induced human osteoclastogenesis and human Th17 differentiation. Human CD14(+) monocytes were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and IL-17. From day 4, tacrolimus was added to these cultures. Osteoclasts were immunohistologically stained for vitronectin receptor 10days later. IL-17 production from activated T cells stimulated with IL-23 was measured by enzyme-linked immunosorbent assay (ELISA). Th17 differentiation from naïve T cells was assayed by flow cytometry. Tacrolimus potently inhibited IL-17-induced osteoclastogenesis from human monocytes and osteoclast activation. Addition of tacrolimus also reduced production of IL-17 in human activated T cells stimulated with IL-23. Interestingly, the population of human IL-17(+)IFN-γ(-) CD4 T cells or IL-17(+)TNF-α(+) CD4 T cells were decreased by adding of tacrolimus. The present study demonstrates that the inhibitory effect of tacrolimus on IL-17-induced osteoclastogenesis from human monocytes. Tacrolimus also inhibited expression of IL-17 or TNF-α by reducing the proportion of Th17, suggesting that therapeutic effect on Th17-associated disease such as RA, inflammatory bowel disease, multiple sclerosis, psoriasis, or allograft rejection. Copyright © 2012 Elsevier Ltd. All rights reserved.

The Interplay of IL-21 and BAFF in the Formation and Maintenance of Human B Cell Memory

PubMed Central

Karnell, Jodi L.; Ettinger, Rachel

2011-01-01

To date, IL-21 stands out as the most influential cytokine for human B cell activation and differentiation. Indeed, when compared to other important B cell tropic cytokines such as IL-2, IL-4, IL-6 and IL-10, IL-21 is clearly the most potent in terms of its ability to influence humoral immune responses in humans. IL-21 has wide reaching actions in determining how B cells will respond to co-stimulation ranging from induction of cell death upon BCR crosslinking to potent induction of class switch recombination and plasma cell differentiation when CD40 molecules are co-engaged. Another crucial B cell factor, exemplified in recent clinical trials, is BAFF/BLys. BAFF plays a critical role in the survival of human B cells and plasma blasts and influences B cell expansion and migration. Recent evidence has shown that IL-21 and BAFF can work in concert to promote and perhaps maintain humoral immunity in humans. Notably, BAFF has the unique ability to substitute for CD40L activities in regard to IL-21-co-stimulation and differentiation of a specific B cell subpopulation located in the human splenic marginal zone. However, and perhaps surprisingly, BAFF signals do not have the capability to override IL-21-driven cell death events when BCR is engaged. In stark contrast, anti-CD40 ligation of B cells co-activated with IL-21 and anti-IgM not only reverses this aforementioned activation-induced cell death, but transforms this death signal into one that drives plasma cell differentiation. Here we will discuss these two critical B cell factors, IL-21 and BAFF, and their distinct and complimentary effects on human B cell responses. PMID:22566888

Granulocyte-Colony Stimulating Factor Receptor, Tissue Factor, and VEGF-R Bound VEGF in Human Breast Cancer In Loco.

PubMed

Wojtukiewicz, Marek Z; Sierko, Ewa; Skalij, Piotr; Kamińska, Magda; Zimnoch, Lech; Brekken, Ralf A; Thorpe, Philip E

2016-01-01

Doxorubicin and docetaxel-based chemotherapy regimens used in breast cancer patients are associated with high risk of febrile neutropenia (FN). Granulocyte colony-stimulating factors (G-CSF) are recommended for both treating and preventing chemotherapy-induced neutropenia. Increased thrombosis incidence in G-CSF treated patients was reported; however, the underlying mechanisms remain unclear. The principal activator of blood coagulation in cancer is tissue factor (TF). It additionally contributes to cancer progression and stimulates angiogenesis. The main proangiogenic factor is vascular endothelial growth factor (VEGF). The aim of the study was to evaluate granulocyte-colony stimulating factor receptor (G-CSFR), tissue factor (TF) expression and vascular endothelial growth factor receptor (VEGF-R) bound VEGF in human breast cancer in loco. G-CSFR, TF and VEGFR bound VEGF (VEGF: VEGFR) were assessed in 28 breast cancer tissue samples. Immunohistochemical (IHC) methodologies according to ABC technique and double staining IHC procedure were employed utilizing antibodies against G-CSFR, TF and VEGF associated with VEGFR (VEGF: VEGFR). Expression of G-CSFR was demonstrated in 20 breast cancer tissue specimens (71%). In 6 cases (21%) the expression was strong (IRS 9-12). Strong expression of TF was observed in all investigated cases (100%). Moreover, expression of VEGF: VEGFR was visualized in cancer cells (IRS 5-8). No presence of G-CSFR, TF or VEGF: VEGFR was detected on healthy breast cells. Double staining IHC studies revealed co-localization of G-CSFR and TF, G-CSFR and VEGF: VEGFR, as well as TF and VEGF: VEGFR on breast cancer cells and ECs. The results of the study indicate that GCSFR, TF and VEGF: VEGFR expression as well as their co-expression might influence breast cancer biology, and may increase thromboembolic adverse events incidence.

Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET).

PubMed Central

Price, M A; Rogers, A E; Treisman, R

1995-01-01

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation. Images PMID:7540136

Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET).

PubMed

Price, M A; Rogers, A E; Treisman, R

1995-06-01

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation.

The receptor tyrosine kinase ERBB4 is expressed in skin keratinocytes and influences epidermal proliferation.

PubMed

Hoesl, Christine; Röhrl, Jennifer M; Schneider, Marlon R; Dahlhoff, Maik

2018-04-01

The epidermal growth factor receptor (EGFR) and associated receptors ERBB2 and ERBB3 are important for skin development and homeostasis. To date, ERBB4 could not be unambiguously identified in the epidermis. The aim of this study was to analyze the ERBB-receptor family with a special focus on ERBB4 in vitro in human keratinocytes and in vivo in human and murine epidermis. We compared the transcript levels of all ERBB-receptors and the seven EGFR-ligands in HaCaT and A431 cells. ERBB-receptor activity was analyzed after epidermal growth factor (EGF) stimulation by Western blot analysis. The location of the receptors was investigated by immunofluorescence in human keratinocytes and skin. Finally, we investigated the function of ERBB4 in the epidermis of skin-specific ERBB4-knockout mice. After EGF stimulation, all ligands were upregulated except for epigen. Expression levels of EGFR were unchanged, but all other ERBB-receptors were down-regulated after EGF stimulation, although all ERBB-receptors were phosphorylated. We detected ERBB4 at mRNA and protein levels in both human epidermal cell lines and in the basal layer of human and murine epidermis. Skin-specific ERBB4-knockout mice revealed a significantly reduced epidermal thickness with a decreased proliferation rate. ERBB4 is expressed in the basal layer of human epidermis and cultured keratinocytes as well as in murine epidermis. Moreover, ERBB4 is phosphorylated in HaCaT cells due to EGF stimulation, and its deletion in murine epidermis affects skin thickness by decreasing proliferation. ERBB4 is expressed in human keratinocytes and plays a role in murine skin homeostasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

PubMed

Haller, K; Ruckes, T; Schmitt, I; Saul, D; Derow, E; Grassmann, R

2000-11-01

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.

Multiple Mechanisms are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodland, Karin D.; Bollinger, Nikki; Ippolito, Danielle L.

2008-11-14

REVIEW ENTIRE DOCUMENT AT: https://pnlweb.pnl.gov/projects/bsd/ERICA%20Manuscripts%20for%20Review/KD%20Rodland%20D7E80/HMEC_transactivation_ms01_15+Figs.pdf ABSTRACT: Using a single nontransformed strain of human mammary epithelial cells, we found that the ability of multiple growth factors and cytokines to induce ERK phosphorylation was dependent on EGFR activity. These included lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factoralpha. In contrast, hepatocyte growth factor could stimulate ERK phosphorylation independent of EGFR activity...

Infrared neural stimulation of human spinal nerve roots in vivo

PubMed Central

Cayce, Jonathan M.; Wells, Jonathon D.; Malphrus, Jonathan D.; Kao, Chris; Thomsen, Sharon; Tulipan, Noel B.; Konrad, Peter E.; Jansen, E. Duco; Mahadevan-Jansen, Anita

2015-01-01

Abstract. Infrared neural stimulation (INS) is a neurostimulation modality that uses pulsed infrared light to evoke artifact-free, spatially precise neural activity with a noncontact interface; however, the technique has not been demonstrated in humans. The objective of this study is to demonstrate the safety and efficacy of INS in humans in vivo. The feasibility of INS in humans was assessed in patients (n=7) undergoing selective dorsal root rhizotomy, where hyperactive dorsal roots, identified for transection, were stimulated in vivo with INS on two to three sites per nerve with electromyogram recordings acquired throughout the stimulation. The stimulated dorsal root was removed and histology was performed to determine thermal damage thresholds of INS. Threshold activation of human dorsal rootlets occurred in 63% of nerves for radiant exposures between 0.53 and 1.23  J/cm2. In all cases, only one or two monitored muscle groups were activated from INS stimulation of a hyperactive spinal root identified by electrical stimulation. Thermal damage was first noted at 1.09  J/cm2 and a 2∶1 safety ratio was identified. These findings demonstrate the success of INS as a fresh approach for activating human nerves in vivo and providing the necessary safety data needed to pursue clinically driven therapeutic and diagnostic applications of INS in humans. PMID:26157986

Activation of human B cells by phosphorothioate oligodeoxynucleotides.

PubMed Central

Liang, H; Nishioka, Y; Reich, C F; Pisetsky, D S; Lipsky, P E

1996-01-01

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors. PMID:8787674

Phospholipase A2 Inhibitor from Crotalus durissus terrificus rattlesnake: Effects on human peripheral blood mononuclear cells and human neutrophils cells.

PubMed

Xavier, Caroline V; da S Setúbal, Sulamita; Lacouth-Silva, Fabianne; Pontes, Adriana S; Nery, Neriane M; de Castro, Onassis Boeri; Fernandes, Carla F C; Soares, Andreimar M; Fortes-Dias, Consuelo L; Zuliani, Juliana P

2017-12-01

Crotalus Neutralizing Factor (CNF) is an inhibitor of phospholipase A 2 (PLA 2 ), present in the blood plasma of Crotalus durissus terrificus snake. This inhibitor neutralizes the lethal and enzymatic activity of crotoxin, the main neurotoxin from this venom. In this study, we investigated the effects of CNF on the functionality of human peripheral blood mononuclear cells (PBMCs) and human neutrophils. The following parameters were evaluated: viability and proliferation, chemotaxis, cytokines and LTB 4 production, cytosolic PLA 2 s activity, myeloperoxidase (MPO) and superoxide anion (O 2 - ) production. CNF showed no toxicity on PBMCs or neutrophils, and acts by stimulating the release of TNF-α and LTB 4 , but neither stimulates IL-10 and IL-2 nor affects PBMCs proliferation and O 2 - release. In neutrophils, CNF induces chemotaxis but does not induce the release of both MPO and O 2 - . However, it induces LTB 4 and IL-8 production. These data show the influence of CNF on PBMCs' function by inducing TNF-α and LTB 4 production, and on neutrophils, by stimulating chemotaxis and LTB 4 production, via cytosolic PLA 2 activity, and IL-8 release. The inflammatory profile produced by CNF is shown for the first time. Our present results suggest that CNF has a role in activation of leukocytes and exert proinflammatory effects on these cell. Copyright © 2017 Elsevier B.V. All rights reserved.

Angiopoietin‐like 4 promotes angiogenesis in the tendon and is increased in cyclically loaded tendon fibroblasts

PubMed Central

Mousavizadeh, Rouhollah; Scott, Alex; Lu, Alex; Ardekani, Gholamreza S; Behzad, Hayedeh; Lundgreen, Kirsten; Ghaffari, Mazyar; McCormack, Robert G

2016-01-01

Key points Angiopoietin‐like 4 (ANGPTL4) modulates tendon neovascularization.Cyclic loading stimulates the activity of transforming growth factor‐β and hypoxia‐inducible factor 1α and thereby increases the expression and release of ANGPTL4 from human tendon cells.Targeting ANGPTL4 and its regulatory pathways is a potential avenue for regulating tendon vascularization to improve tendon healing or adaptation. Abstract The mechanisms that regulate angiogenic activity in injured or mechanically loaded tendons are poorly understood. The present study examined the potential role of angiopoietin‐like 4 (ANGPTL4) in the angiogenic response of tendons subjected to repetitive mechanical loading or injury. Cyclic stretching of human tendon fibroblasts stimulated the expression and release of ANGPTL4 protein via transforming growth factor‐β (TGF‐β) and hypoxia‐inducible factor 1α (HIF‐1α) signalling, and the released ANGPTL4 was pro‐angiogenic. Angiogenic activity was increased following ANGPTL4 injection into mouse patellar tendons, whereas the patellar tendons of ANGPTL4 knockout mice displayed reduced angiogenesis following injury. In human rotator cuff tendons, the expression of ANGPTL4 was correlated with the density of tendon endothelial cells. To our knowledge, this is the first study characterizing a role of ANGPTL4 in the tendon. ANGPTL4 may assist in the regulation of vascularity in the injured or mechanically loaded tendon. TGF‐β and HIF‐1α comprise two signalling pathways that modulate the expression of ANGPTL4 by mechanically stimulated tendon fibroblasts and, in the future, these could be manipulated to influence tendon healing or adaptation. PMID:26670924

Hematopoietic growth factors and human acute leukemia.

PubMed

Löwenberg, B; Touw, I

1988-10-22

The study of myelopoietic maturation arrest in acute myeloblastic leukemia (AML) has been eased by availability of the human recombinant hemopoietic growth factors, macrophage colony stimulating factor (M-CSF), granulocyte-(G-CSF), granulocyte-macrophage-(GM-CSF) and multilineage stimulating factor (IL-3). Nonphysiological expansion of the leukemic population is not due to escape from control by these factors. Proliferation in vitro of AML cells is dependent on the presence of one or several factors in most cases. The pattern of factor-dependency does not correlate with morphological criteria in individual cases, and may thus offer a new tool for classification of AML. Overproduction of undifferentiated cells is not due to abnormal expression of receptors for the stimulating factors acting at an immature level. Rather, autocrine secretion of early acting lymphokines maintains proliferation of the leukemic clone. When looking at causes of leukemic dysregulation, yet undefined inhibitors of differentiation probably are of equal importance as dysequilibrated stimulation by lymphokines.

Manifestation of cryptic fibroblast tissue factor occurs at detergent concentrations which dissolve the plasma membrane.

PubMed

Carson, S D

1996-04-01

Cultured fibroblasts treated with increasing concentrations of detergents expressed only encrypted levels of tissue factor activity (measured by fX activation in the presence of fVIIa), characteristic of undamaged cells, until each detergent reached a critical concentration at which the cryptic tissue factor activity was manifested. Beyond the narrow ranges of concentrations over which the detergents stimulated tissue factor activity, the detergents were inhibitory. Studies with Triton X-100 and octyl glucoside revealed that manifestation of tissue factor activity coincided with breakdown of the plasma membrane. The magnitude of the increased tissue factor activity differed among detergents, with octyl glucoside giving the largest response. The tissue factor that was active after Triton X-100 treatment remained mostly associated with the insoluble cell residue, whereas the concentration of octyl glucoside which stimulated activity released tissue factor activity into the supernatant. Radiolabeled antibody against human tissue factor was used to show that a small percentage of the total accessible tissue factor remained in the insoluble fraction after treatment with either non-ionic detergent. Chromatographic analysis of lipids extracted from cells treated with detergents and dansyl chloride showed dansyl-reactivity of phosphatidylserine on intact cells, and solubilization of membrane lipids at sublytic concentrations of detergents. These findings reveal that there is a critical level of detergent-induced membrane damage at which tissue factor activity is maximally expressed, in essentially an all-or-none manner. The results are consistent with a major role for phospholipid asymmetry in regulation of tissue factor specific activity, but require either maintenance of asymmetry during sublytic detergent perturbation of the plasma membrane or additional control mechanisms.

Synergistic regulation of the human interleukin-12 p40 promoter by NFkappaB and Ets transcription factors in Epstein-Barr virus-transformed B cells and macrophages.

PubMed

Gri, G; Savio, D; Trinchieri, G; Ma, X

1998-03-13

Monocytes/macrophages produce interleukin-12 (IL-12) in response to pathogenic stimulation, whereas most Epstein-Barr virus-transformed (EBV+) B cells constitutively secrete IL-12. The molecular mechanism regulating the constitutive IL-12 gene expression in EBV+ B cells has not been addressed. In this study, using the EBV+ B cell line RPMI-8866, we localized to the human IL-12 p40 promoter two essential cis elements, the NFkappaB site and the Ets site. The NFkappaB site was shown to interact with members of the NFkappaB family: p50 and c-Rel. The Ets site constitutively bound a multi-component Ets-2-containing complex. While the NFkappaB and Ets sites appear equally critical for inducible p40 promoter activity in macrophage cell lines, NFkappaB plays a more dominant role in the constitutive p40 promoter activity in EBV+ B cells. Transient expression of Ets-2 and c-Rel in B, T, and monocytic cell lines synergistically activated the IL-12 p40 promoter, apparently overcoming the requirement for cell type- or stimulant-specific transcription factors. These data provide new evidence that full activation of the human IL-12 p40 promoter may result primarily from the interplay between NFkappaB and Ets family members.

Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

PubMed

El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

2018-01-17

Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

Plasmodium circumsporozoite protein suppresses the growth of A549 cells via inhibiting nuclear transcription factor κB.

PubMed

Deng, Xu-Feng; Zhou, Dong; Liu, Quan-Xing; Zheng, Hong; Ding, Yan; Xu, Wen-Yue; Min, Jia-Xin; Dai, Ji-Gang

2018-05-01

Blocking the activation of nuclear factor κB (NF-κB) is a promising strategy for the treatment of non-small cell lung cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was previously reported to block NF-κB activation in hepatocytes. Therefore, in the present study, the effect of CSP on the growth of the human lung cancer cell line, A549, was investigated. It was demonstrated that transfection with a recombinant plasmid expressing CSP was able to inhibit the proliferation of A549 cells in a dose-dependent manner and induce the apoptosis of A549 cells. A NF-κB gene reporter assay indicated that CSP and its nuclear localization signal (NLS) motif were able to equally suppress the activation of NF-κB following stimulation with human recombinant tumor necrosis factor (TNF)-α in A549 cells. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but was able to markedly inhibit the nuclear translocation of NF-κB in TNF-α stimulated A549 cells. Therefore, the data suggest that CSP may be investigated as a potential novel NF-κB inhibitor for the treatment of lung cancer.

Thyroid hormone stimulates progesterone release from human luteal cells by generating a proteinaceous factor.

PubMed

Datta, M; Roy, P; Banerjee, J; Bhattacharya, S

1998-09-01

Blood samples collected from 29 women (aged between 19 and 35 years) during the luteal phase of the menstrual cycle (between days 18 and 23 of the cycle) showed that deficiency in thyroid hormone level is related to a decrease in progesterone (P4) secretion. To observe the effect of thyroid hormone on human ovarian luteal cells, 3,5,3'-triiodothyronine (T3; 125 ng/ml) was added to luteal cells in vitro. T3 significantly stimulated progesterone release (P < 0.01) from luteal cells and this could be blocked by cycloheximide, indicating a protein mediator for the T3 effect. The T3 stimulatory effect was inhibited by anti-T3 antibody suggesting specificity of T3 action. Addition of T3 caused a more than threefold increase in cellular protein synthesis which was inhibited by cycloheximide. Preparation of partially purified thyroid hormone-induced factor (TIF) (from peak II of Sephadex G 100 chromatography of T3-incubated cells), and its addition to luteal cell incubations caused a significant increase in P4 release (P < 0.05). Incubation with trypsin or treatment with heat destroyed the stimulatory effect of TIF on P4 release, indicating the proteinaceous nature of TIF. Purified thyroid hormone-induced protein. (TIP) from rat granulosa cells and fish ovarian follicles greatly stimulated P4 release from human luteal cells. These results suggest that T3 stimulation of P4 release from human luteal cells is not direct, but is mediated through a putative protein factor, which appears to be a protein conserved through evolution as far as its biological activity is concerned.

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed Central

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-01-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets. PMID:10947961

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

PubMed

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Further Characterization of an Interleukin-2-1Ike Cytokine Produced by Xenopus Laevis T Lymphocytes

PubMed Central

Haynes, Laura

1993-01-01

A T-cell growth factor (TCGF) is produced by antigen- or mitogen-stimulated T lymphocytes from the South African clawed frog Xenopus laevis. This study further defines the physical and biological properties of this cytokine and demonstrates that TCGF is biochemically similar to mammalian interleukin-2 (IL-2). Biologically active TCGF eluted from SDS-PAGE displays a Mr of 16 kD and lectin-affinity chromatography indicates that the three-dimensionmal configuration of carbohydrates on TCGF and human IL-2 is similar. Secretion of TCGF is detectable 1 day after stimulation of splenocytes with the T-cell mitogen phytohemagglutinin (PHA) and peaks following 2 to 3 days of stimulation. Finally, despite the biological and physical similarities between Xenopus TCGF and mammalian IL-2, anti-human IL-2 monoclonal antibodies do not recognize Xenopus TCGF. PMID:8281036

3,4,5-Tricaffeoylquinic acid inhibits tumor necrosis factor-α-stimulated production of inflammatory mediators in keratinocytes via suppression of Akt- and NF-κB-pathways.

PubMed

Lee, Chung Soo; Lee, Seon Ae; Kim, Yun Jeong; Seo, Seong Jun; Lee, Min Won

2011-11-01

Keratinocytes may play an important role in the pathogenesis of skin disease in atopic dermatitis. Caffeoyl derivatives are demonstrated to have anti-inflammatory and anti-oxidant effects. However, the effect of 3,4,5-tricaffeoylquinic acid prepared from Aconium koreanum on the pro-inflammatory cytokine-stimulated keratinocyte responses remains uncertain. In human keratinocytes, we investigated the effect of 3,4,5-tricaffeoylquinic acid on the tumor necrosis factor (TNF)-α-stimulated production of inflammatory mediators in relation to the nuclear factor (NF)-κB and cell signaling Akt, which regulates the transcription genes involved in immune and inflammatory responses. 3,4,5-Tricaffeoylquinic acid inhibited the TNF-α-stimulated production of cytokines (IL-1β and IL-8) and chemokine (CCL17 and CCL27) in keratinocytes. Bay 11-7085 (an inhibitor of NF-κB activation) and Akt inhibitor attenuated the TNF-α-induced formation of inflammatory mediators. 3,4,5-Tricaffeoylquinic acid, Bay 11-7085, Akt inhibitor and N-acetylcysteine inhibited the TNF-α-induced activation of NF-κB, activation of Akt, and formation of reactive oxygen and nitrogen species. The results show that 3,4,5-tricaffeoylquinic acid seems to attenuate the TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of Akt and NF-κB pathways which may be mediated by reactive oxygen species. The findings suggest that 3,4,5-tricaffeoylquinic acid may exert an inhibitory effect against the pro-inflammatory mediator-induced skin disease. Copyright © 2011 Elsevier B.V. All rights reserved.

Anti- Schistosomular Activity of Human Monocytes/Macrophages in Response to Interleukin-3 and Granulocyte-Macrophage Colonystimulating Factor Stimulation

PubMed Central

Nissimov, L.; Lengy, J.; Keisari, Y.

1994-01-01

Human monocytes, co-incubated for 7 days in culture with GM-CSF or IL-3 but not with IFN-γ, exerted a variable schistosotnulicidal effect on Schistosoma mansoni parasites when grown in 96-well round-bottomed plates but not in flat-bottomed plates. Addition of LPS or IFN-γ or both, for the last 48 h did not enhance the cidal effect. Addition of LPS but not IFN-γ to the pre-incubated cells with GM-CSF or IL-3 markedly stimulated TNF-α production by the cells but not their cidal activity. The variable cidal effects obtained with the monocytes/macrophages from different donors suggest that these effects may be genetically predetermined and are possibly linked to blood group markers or to MHC class I or II antigens. PMID:18475576

Human Dental Pulp Stem Cells Suppress Alloantigen-induced Immunity by Stimulating T Cells to Release Transforming Growth Factor Beta.

PubMed

Kwack, Kyu Hwan; Lee, Jung Min; Park, Sang Hyuk; Lee, Hyeon Woo

2017-01-01

Human dental pulp stem cells (hDPSCs) are ideal candidates for regenerating damaged dental tissue. To examine the possibility that hDPSCs may be used to regenerate pulp, we tested their in vitro effects on acute allogeneic immune responses. A peripheral blood mononuclear cell (PBMC) proliferation assay and immunoglobulin (Ig) production assay were performed to evaluate the immunosuppressive properties of hDPSCs. The mixed lymphocyte reaction was suppressed by incubation with hDPSCs. Transforming growth factor beta (TGF-β) was the major soluble factor responsible for inhibiting the allogeneic proliferation of PBMCs. The production of IgM and IgG by allogeneic activation of responder B lymphocytes was also completely abrogated by TGF-β released from hDPSCs via interferon gamma in response to activation of the responder T lymphocytes. hDPSCs inhibit acute allogeneic immune responses by their release of TGF-β as a result of allogeneic stimulation of T lymphocytes. This study provides an insight into the potential clinical use of hDPSCs for allogeneic transplantation. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo.

PubMed Central

Taub, D D; Anver, M; Oppenheim, J J; Longo, D L; Murphy, W J

1996-01-01

IL-8 has been shown to be a human neutrophil and T cell chemoattractant in vitro. In an effort to assess the in vivo effects of IL-8 on human leukocyte migration, we examined the ability of rhIL-8 to induce human T cell infiltration using a human/mouse model in which SCID mice were administered human peripheral blood lymphocytes intraperitoneally, followed by subcutaneous injections of rhIL-8. rhIL-8 induced predominantly murine neutrophil accumulation by 4 h after administration while recombinant human macrophage inflammatory protein-1beta (rhMIP-1beta) induced both murine monocytes and human T cell infiltration during the same time period as determined by immunohistology. Interestingly, 72 h after chemokine administration, a marked human T cell infiltrate was observed in the IL-8 injection site suggesting that rhIL-8 may be acting indirectly possibly through a murine neutrophil-derived T cell chemoattractant. This hypothesis was confirmed using granulocyte-depleted SCID mice. Moreover, human neutrophils stimulated in vitro with IL-8 were found to release granule-derived factor(s) that induce in vitro T cell and monocyte chemotaxis and chemokinesis. This T cell and monocyte chemotactic activity was detected in extracts of both azurophilic and specific granules. Together, these results demonstrate that neutrophils store and release, upon stimulation with IL-8 or other neutrophil activators, chemoattractants that mediate T cell and monocyte accumulation at sites of inflammation. PMID:8621778

Adenosine Triphosphate stimulates differentiation and mineralization in human osteoblast-like Saos-2 cells.

PubMed

Cutarelli, Alessandro; Marini, Mario; Tancredi, Virginia; D'Arcangelo, Giovanna; Murdocca, Michela; Frank, Claudio; Tarantino, Umberto

2016-05-01

In the last years adenosine triphosphate (ATP) and subsequent purinergic system activation through P2 receptors were investigated highlighting their pivotal role in bone tissue biology. In osteoblasts ATP can regulate several activities like cell proliferation, cell death, cell differentiation and matrix mineralization. Since controversial results exist, in this study we analyzed the ATP effects on differentiation and mineralization in human osteoblast-like Saos-2 cells. We showed for the first time the altered functional activity of ATP receptors. Despite that, we found that ATP can reduce cell proliferation and stimulate osteogenic differentiation mainly in the early stages of in vitro maturation as evidenced by the enhanced expression of alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and Osteocalcin (OC) genes and by the increased ALP activity. Moreover, we found that ATP can affect mineralization in a biphasic manner, at low concentrations ATP always increases mineral deposition while at high concentrations it always reduces mineral deposition. In conclusion, we show the osteogenic effect of ATP on both early and late stage activities like differentiation and mineralization, for the first time in human osteoblastic cells. © 2016 Japanese Society of Developmental Biologists.

Suppression of bactericidal activity of human polymorphonuclear leukocytes by Bacteroides gingivalis.

PubMed Central

Yoneda, M; Maeda, K; Aono, M

1990-01-01

The direct effects of the culture supernatant of oral microorganisms on the bactericidal activity of human polymorphonuclear leukocytes (PMNs) were investigated. The bactericidal activity of PMNs, which were preincubated with the supernatant of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides melaninogenicus or phosphate-buffered saline, was examined by counting the surviving bacteria. B. gingivalis-treated PMNs were found to have a diminished ability for killing bacteria in the presence or absence of serum. The chemiluminescence response of PMNs, which were preincubated with the culture supernatant of B. gingivalis, was strikingly reduced compared with that of PMNs preincubated with phosphate-buffered saline or other bacterial culture supernatants. The production of superoxide anion (O2-) by PMNs stimulated with either formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate was reduced in both cases after the PMNs were preincubated with the culture supernatant of B. gingivalis. However, it was observed that there was more reduction in superoxide anion (O2-) production stimulated with formyl-methionyl-leucyl-phenylalanine compared with that stimulated with phorbol myristate acetate. These results suggest that B. gingivalis releases a factor which interferes with the bactericidal activity of PMNs by modulating the generation of reactive oxygen species. These suppressive effects on bactericidal activity may be important in the pathogenesis of this microorganism. PMID:2153632

Methylquercetins stimulate melanin biosynthesis in a three-dimensional skin model.

PubMed

Yamauchi, Kosei; Mitsunaga, Tohru

2018-03-01

In a previous study, we found that both synthetic 3-O-methylquercetin (3MQ) and 3,4',7-O-trimethylquercetin (34'7TMQ) increased extracellular melanin content. 34'7TMQ increased the activity of melanogenic enzymes by stimulating the p38 pathway and the expression of microphthalmia-associated transcription factor (MITF). In contrast, 3MQ increased the activity of melanogenic enzymes without the involvement of MITF, which suggests that 3MQ inhibits the degradation of melanogenic enzymes. In the present study, we investigated the effects of 3MQ and 34'7TMQ on melanogenesis in normal human melanocytes and using a commercial three-dimensional (3D) skin model system. Both 3MQ and 34'7TMQ elongated the dendrites of normal human melanocytes from a Caucasian donor, but did not stimulate melanogenesis in the melanocytes. In the 3D skin model, which included melanocytes from an Asian donor, 3MQ and 34'7TMQ increased and elongated the melanocytes and showed a tendency to stimulate melanogenesis. These results suggest that 3MQ and 34'7TMQ could be put to practical use in skin care products and agents aimed at preventing hair graying.

HCG-Activated Human Peripheral Blood Mononuclear Cells (PBMC) Promote Trophoblast Cell Invasion

PubMed Central

Wang, Yaqin; Guo, Yue; Zhou, Danni; Xu, Mei; Ding, Jinli; Yang, Jing

2015-01-01

Successful embryo implantation and placentation depend on appropriate trophoblast invasion into the maternal endometrial stroma. Human chorionic gonadotropin (hCG) is one of the earliest embryo-derived secreted signals in the peripheral blood mononuclear cells (PBMC) that abundantly expresses hCG receptors. The aims of this study were to estimate the effect of human embryo–secreted hCG on PBMC function and investigate the role and underlying mechanisms of activated PBMC in trophoblast invasion. Blood samples were collected from women undergoing benign gynecological surgery during the mid-secretory phase. PBMC were isolated and stimulated with or without hCG for 0 or 24 h. Interleukin-1β (IL-1β) and leukemia inhibitory factor (LIF) expressions in PBMC were detected by enzyme-linked immunosorbent assay and real-time polymerase chain reaction (PCR). The JAR cell line served as a model for trophoblast cells and was divided into four groups: control, hCG only, PBMC only, and PBMC with hCG. JAR cell invasive and proliferative abilities were detected by trans-well and CCK8 assays and matrix metalloproteinase (MMP)-2 (MMP-2), MMP-9, vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 expressions in JAR cells were detected by western blotting and real-time PCR analysis. We found that hCG can remarkably promote IL-1β and LIF promotion in PBMC after 24-h culture. PBMC activated by hCG significantly increased the number of invasive JAR cells in an invasion assay without affecting proliferation, and hCG-activated PBMC significantly increased MMP-2, MMP-9, and VEGF and decreased TIMP-1 and TIMP-2 expressions in JAR cells in a dose-dependent manner. This study demonstrated that hCG stimulates cytokine secretion in human PBMC and could stimulate trophoblast invasion. PMID:26087261

Vestibular influences on autonomic cardiovascular control in humans

NASA Technical Reports Server (NTRS)

Biaggioni, I.; Costa, F.; Kaufmann, H.; Robertson, D. (Principal Investigator)

1998-01-01

There is substantial evidence that anatomical connections exist between vestibular and autonomic nuclei. Animal studies have shown functional interactions between the vestibular and autonomic systems. The nature of these interactions, however, is complex and has not been fully defined. Vestibular stimulation has been consistently found to reduce blood pressure in animals. Given the potential interaction between vestibular and autonomic pathways this finding could be explained by a reduction in sympathetic activity. However, rather than sympathetic inhibition, vestibular stimulation has consistently been shown to increase sympathetic outflow in cardiac and splanchnic vascular beds in most experimental models. Several clinical observations suggest that a link between vestibular and autonomic systems may also exist in humans. However, direct evidence for vestibular/autonomic interactions in humans is sparse. Motion sickness has been found to induce forearm vasodilation and reduce baroreflex gain, and head down neck flexion induces transient forearm and calf vasoconstriction. On the other hand, studies using optokinetic stimulation have found either very small, variable, or inconsistent changes in heart rate and blood pressure, despite substantial symptoms of motion sickness. Furthermore, caloric stimulation severe enough to produce nystagmus, dizziness, and nausea had no effect on sympathetic nerve activity measured directly with microneurography. No effect was observed on heart rate, blood pressure, or plasma norepinephrine. Several factors may explain the apparent discordance of these results, but more research is needed before we can define the potential importance of vestibular input to cardiovascular regulation and orthostatic tolerance in humans.

An enkephalin-like molecule in earthworm coelomic fluid modifies leukocyte behavior.

PubMed

Cooper, E L; Leung, M K; Suzuki, M M; Vick, K; Cadet, P; Stefano, G B

1993-01-01

Substances that were immunoreactive in an RIA specific for met-enkephalin were detected following HPLC fractionation of earthworm coelomic fluid. Earthworm coelomocytes and human granulocytes were analyzed for changes in conformation based on measurements of cellular area and perimeter and expressed mathematically by using the Form Factor (FF). For coelomocytes the FF decreased following exposure to DAMA, a synthetic enkephalin analogue (D-Ala2, Met5-enkephalinamide). DAMA stimulated migration whereas untreated cells and those exposed to the specific opiate blocker naloxone did not move. The enkephalin-like molecule when exposed to human granulocytes stimulated an increased number of activated cells. Our results suggest a relationship between the immune and nervous systems of earthworms.

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

PubMed Central

Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

2014-01-01

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

PubMed Central

Lindquist, Jonathan A.; Arhel, Nathalie; Felder, Edward; Karl, Sabine; Haas, Tobias L.; Fulda, Simone; Walczak, Henning; Kirchhoff, Frank; Debatin, Klaus-Michael

2009-01-01

CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion. PMID:19487421

Up-regulation of IL-23 expression in human dental pulp fibroblasts by IL-17 via activation of the NF-κB and MAPK pathways.

PubMed

Wei, L; Liu, M; Xiong, H; Peng, B

2017-11-06

To investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-17 on HDPFs-mediated IL-23 production and the molecular mechanism involved. Interleukin (IL)-17R expression was determined by semi-quantitative reverse transcriptase-polymerase chain reaction and Western blot in cultured human dental pulp fibroblasts (HDPFs). Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-23 mRNA and protein levels in IL-17-stimulated HDPFs, respectively. The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways that mediate the IL-17-stimulated production of IL-23 was investigated using Western blot and specific signalling inhibitor analyses. Statistical analyses were performed using Kruskal-Wallis tests followed by the Mann-Whitney U-test. Statistical significance was considered when the P value < 0.05. Primary HDPFs steadily expressed IL-17R mRNA and surface-bound protein. IL-17 stimulated the expression of IL-23 mRNA and protein in cultured human dental pulp fibroblasts, which was attenuated by IL-17 or IL-17R neutralizing antibodies. In accordance with the enhanced expression of IL-23, IL-17 stimulation resulted in rapid activation of p38 MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK) and NF-κB in HDPFs. Inhibitors of p38 MAPK, ERK 1/2 or NF-κB significantly suppressed, whereas blocking JNK substantially augmented IL-23 production from IL-17-stimulated HDPFs. HDPFs expressed IL-17R and responded to IL-17 to produce IL-23 via the activation of the NF-κB and MAPK signalling pathways. The findings provide insights into the cellular mechanisms of the participation of IL-17 in the activation of HDPFs in inflamed pulp tissue. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis

PubMed Central

Vara, Dina; Watt, Joanna M.; Fortunato, Tiago M.; Mellor, Harry; Burgess, Matthew; Wicks, Kate; Mace, Kimberly; Reeksting, Shaun; Lubben, Anneke; Wheeler-Jones, Caroline P.D.

2018-01-01

Abstract Aims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2−/− mice. Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130. PMID:28793782

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

PubMed

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cortisol and 3,4-Methylenedioxymethamphetamine: Neurohormonal Aspects of Bioenergetic Stress in Ecstasy Users

PubMed Central

Parrott, A.C.

2009-01-01

Aims 3,4-Methylenedioxymethamphetamine (MDMA) can affect both neurotransmitter and neurohormonal activity. This review will debate the role of the metabolic activation hormone cortisol for the psychobiological effects of ecstasy/MDMA. Methods The empirical literature on cortisol release following acute MDMA administration and cortisol functioning in drug-free recreational ecstasy/MDMA users will be reviewed. This will be followed by an overview of cortisol as a bioenergetic stress neurohormone, and a debate on how it could be modulating the acute and chronic psychobiological effects of MDMA. Results Cortisol release is increased by stimulatory factors, including physical activity, thermal stress and stimulant drugs. In laboratory studies MDMA leads to an acute cortisol increase of around 150% in sedentary humans. In MDMA-using dance clubbers, the cortisol levels are increased by around 800%, possibly due to the combined factors of stimulant drug, physical exertion and psychosocial stimulation. Regular ecstasy/MDMA users also demonstrate changes in baseline cortisol levels and cortisol reactivity, with compromised hypothalamic-pituitary-adrenal activity. Nonpharmacological research has shown how cortisol is important for psychological aspects such as memory, cognition, sleep, impulsivity, depression and neuronal damage. These same functions are often impaired in recreational ecstasy/MDMA users, and cortisol may be an important modulatory co-factor. Conclusions The energizing hormone cortisol is involved in the psychobiology of MDMA, probably via its effects on energy metabolism. Acute cortisol release may potentiate the stimulating effects of MDMA in dance clubbers. Chronically, cortisol may contribute to the variance in functional and structural consequences of repeated ecstasy usage. PMID:19893332

Inhibition of interferon-gamma expression by osmotic shrinkage of peripheral blood lymphocytes.

PubMed

Lang, K S; Weigert, C; Braedel, S; Fillon, S; Palmada, M; Schleicher, E; Rammensee, H-G; Lang, F

2003-01-01

A hypertonic environment, as it prevails in renal medulla or in hyperosmolar states such as hyperglycemia of diabetes mellitus, has been shown to impair the immune response, thus facilitating the development of infection. The present experiments were performed to test whether hypertonicity influences activation of T lymphocytes. To this end, peripheral blood lymphocytes (PBL) of cytomegalovirus (CMV)-positive donors were stimulated by human leukocyte antigen (HLA)-A2-restricted CMV epitope NLVPMVATV to produce interferon (IFN)-gamma at varying extracellular osmolarity. As a result, increasing extracellular osmolarity during exposure to the CMV antigen indeed decreased IFN-gamma formation. Addition of NaCl was more effective than urea. A 50% inhibition was observed at 350 mosM by addition of NaCl. The combined application of the Ca(2+) ionophore ionomycin (1 microg/ml) and the phorbol ester phorbol 12-myristate 13-acetate (PMA; 5 microg/ml) stimulated IFN-gamma production, an effect again reversed by hyperosmolarity. Moreover, hyperosmolarity abrogated the stimulating effect of ionomycin (1 microg/ml) and PMA (5 microg/ml) on the transcription factors activator protein (AP)-1, nuclear factor of activated T cells (NFAT), and NF-kappaB but not Sp1. In conclusion, osmotic cell shrinkage blunts the stimulatory action of antigen exposure on IFN-gamma production, an effect explained at least partially by suppression of transcription factor activation.

Insulin signalling mechanisms for triacylglycerol storage.

PubMed

Czech, M P; Tencerova, M; Pedersen, D J; Aouadi, M

2013-05-01

Insulin signalling is uniquely required for storing energy as fat in humans. While de novo synthesis of fatty acids and triacylglycerol occurs mostly in liver, adipose tissue is the primary site for triacylglycerol storage. Insulin signalling mechanisms in adipose tissue that stimulate hydrolysis of circulating triacylglycerol, uptake of the released fatty acids and their conversion to triacylglycerol are poorly understood. New findings include (1) activation of DNA-dependent protein kinase to stimulate upstream stimulatory factor (USF)1/USF2 heterodimers, enhancing the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c); (2) stimulation of fatty acid synthase through AMP kinase modulation; (3) mobilisation of lipid droplet proteins to promote retention of triacylglycerol; and (4) upregulation of a novel carbohydrate response element binding protein β isoform that potently stimulates transcription of lipogenic enzymes. Additionally, insulin signalling through mammalian target of rapamycin to activate transcription and processing of SREBP1c described in liver may apply to adipose tissue. Paradoxically, insulin resistance in obesity and type 2 diabetes is associated with increased triacylglycerol synthesis in liver, while it is decreased in adipose tissue. This and other mysteries about insulin signalling and insulin resistance in adipose tissue make this topic especially fertile for future research.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mu, Shumin; Hospital Affiliated to Shandong Traditional Chinese Medicine University, Jinan 250011; Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021

Highlights: Black-Right-Pointing-Pointer Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. Black-Right-Pointing-Pointer Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. Black-Right-Pointing-Pointer Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exceptionmore » of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.« less

Highly efficient Cas9-mediated transcriptional programming

DOE PAGES

Chavez, Alejandro; Scheiman, Jonathan; Vora, Suhani; ...

2015-03-02

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. Here we describe an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. Here, we demonstrate its utility in activating endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promotermore » activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine displayed enhanced invasiveness. ► Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ► MAPK/AP-1 and ROS/NF-κB signals are involved in nicotine-induced uPAR.« less

Biologic properties of the thymocyte-activating factor (CETAF) produced by a rabbit corneal cell line (SIRC).

PubMed

Grabner, G; Luger, T A; Luger, B M; Smolin, G; Oh, J O

1983-05-01

Rabbit corneal epithelial cell cultures produce a cytokine (CETAF) that greatly enhances the proliferation of C3H/HeJ mouse thymocytes. The rabbit corneal cell line SIRC was used to generate CETAF activity in the culture supernatant. CETAF was then partially purified by Sephacryl S-200 gel filtration, where peaks of activity eluted in a molecular weight range of 95,000-55,000 (CETAF I) and 30,000-15,000 (CETAF II). Similar to the epidermal cell-derived thymocyte-activating factor (ETAF), CETAF (I and II) stimulated the growth of a human dermal fibroblast line (CRL 1445) in a dose-dependent manner, but failed to enhance the proliferation of an Interleukin 2 (IL 2)-dependent T-cell line (CT 6). Although CETAF did not exhibit any IL 2 activity, it clearly enhanced the IL 2 production by C3H/HeJ mouse splenocytes stimulated with suboptimal doses of lectins. Crude SIRC supernatants as well as the partially purified CETAF preparations showed a marked inhibition of polymorphonuclear neutrophil migration at high concentrations, but were significantly chemotactic when diluted samples were tested. CETAF release by SIRC cells was increased by stimulation with mitomycin C, phorbolmyristate acetate, hydroxyurea, silica, lipopolysaccaride B, and when the cells were cultured under serum-free conditions. These observations suggest that corneal epithelial cells may not only interact with the immune system in a way similar to keratinocytes, but may also stimulate corneal stromal cell through the production of CETAF.

Effects of globularifolin on cell survival, nuclear factor-κB activity, neopterin production, tryptophan breakdown and free radicals in vitro.

PubMed

Sipahi, Hande; Becker, Kathrin; Gostner, Johanna M; Charehsaz, Mohammad; Kirmizibekmez, Hasan; Schennach, Harald; Aydin, Ahmet; Fuchs, Dietmar

2014-01-01

The potential effects of globularifolin, an acylated iridoid glucoside, on cell survival, inflammation markers and free radicals scavenging were investigated. Viability assay on human myelomomonocytic cell line THP-1 and human peripheral blood mononuclear cells (PBMC) using the Cell-Titer Blue assay proved that globularifolin had no toxic effect at the tested concentrations. Conversely, it is proportional to the dose globularifolin increased growth of THP-1 cells (p <0.01). On human PBMC, globularifolin at 6.25 and 12.5 μM concentrations showed a stimulatory effect, while at 12.5-200 μM it suppressed response of PBMC to stimulation with phytohemagglutinin (PHA). Globularifolin (50-200 μM) enhanced neopterin formation dose-dependently, whereas tryptophan breakdown was not influenced. At 50-200 μM in unstimulated PBMC in THP-1 cells, globularifolin induced a significant expression of nuclear factor-κB (NF-κB) as was quantified by Quanti-Blue assay. By contrast, in lipopolysaccharide (LPS)-stimulated cells, the higher concentrations of globularifolin suppressed NF-κB expression dose-dependently and a significant decrease was observed at 200 μM concentration. A positive correlation was found between increased neopterin and NF-κB activity (p <0.01). Similarly, a positive correlation was observed between neopterin levels in mitogen-induced cells and NF-κB activity in LPS-stimulated cells after treatment with globularifolin (p=0.001). The free radical scavenging capacity of globularifolin evaluated by Oxygen Radical Absorbance Capacity (ORAC) assay showed relative ORAC values of 0.36±0.05 μmol Trolox equivalent/μmol. All together, results show that natural antioxidant globularifolin might represent a potential immunomodulatory as well as proliferative agent, which deserves further in vitro and in vivo studies. © 2013.

The proangiogenic phenotype of tumor-derived endothelial cells is reverted by the overexpression of platelet-activating factor acetylhydrolase.

PubMed

Doublier, Sophie; Ceretto, Monica; Lupia, Enrico; Bravo, Stefania; Bussolati, Benedetta; Camussi, Giovanni

2007-10-01

We previously reported that human tumor-derived endothelial cells (TEC) have an angiogenic phenotype related to the autocrine production of several angiogenic factors. The purpose of the present study was to evaluate whether an enhanced synthesis of platelet-activating factor (PAF) might contribute to the proangiogenic characteristics of TEC and whether its inactivation might inhibit angiogenesis. To address the potential role of PAF in the proangiogenic characteristics of TEC, we engineered TEC to stably overexpress human plasma PAF-acetylhydrolase (PAF-AH), the major PAF-inactivating enzyme, and we evaluated in vitro and in vivo angiogenesis. TECs were able to synthesize a significantly enhanced amount of PAF compared with normal human microvascular endothelial cells when stimulated with thrombin, vascular endothelial growth factor, or soluble CD154. Transfection of TEC with PAF-AH (TEC-PAF-AH) significantly inhibited apoptosis resistance and spontaneous motility of TEC. In addition, PAF and vascular endothelial growth factor stimulation enhanced the motility and adhesion of TEC but not of TEC-PAF-AH. In vitro, TEC-PAF-AH lost the characteristic ability of TEC to form vessel-like structures when plated on Matrigel. Finally, when cells were injected s.c. within Matrigel in severe combined immunodeficiency mice or coimplanted with a renal carcinoma cell line, the overexpression of PAF-AH induced a significant reduction of functional vessel formation. These results suggest that inactivation of PAF, produced by TEC, by the overexpression of plasma PAF-AH affects survival, migration, and the angiogenic response of TEC both in vitro and in vivo.

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell-cell interaction with activated cells.

PubMed

Okamoto, Takayuki; Akita, Nobuyuki; Hayashi, Tatsuya; Shimaoka, Motomu; Suzuki, Koji

2014-10-01

Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Interleukin-6 production by human monocytes stimulated with Cryptococcus neoformans components.

PubMed Central

Delfino, D; Cianci, L; Lupis, E; Celeste, A; Petrelli, M L; Curró, F; Cusumano, V; Teti, G

1997-01-01

In order to ascertain if Cryptococcus neoformans components can induce interleukin-6 (IL-6) production, we stimulated human whole blood with purified capsular products. Their potencies in stimulating IL-6 release were mannoproteins > galactoxylomannan = glucuronoxylomannan > alpha(1-3)glucan. IL-6 production was tumor necrosis factor alpha independent and required the presence of monocytes and plasma. Since IL-6 can stimulate replication of the human immunodeficiency virus in monocytic cells, these findings may be clinically relevant. PMID:9169790

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

PubMed

González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

2017-10-10

The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

Leishmania major Infection Activates NF-κB and Interferon Regulatory Factors 1 and 8 in Human Dendritic Cells▿

PubMed Central

Jayakumar, Asha; Donovan, Michael J.; Tripathi, Vinita; Ramalho-Ortigao, Marcelo; McDowell, Mary Ann

2008-01-01

The salient feature of dendritic cells (DC) is the initiation of appropriate adaptive immune responses by discriminating between pathogens. Using a prototypic model of intracellular infection, we previously showed that Leishmania major parasites prime human DC for efficient interleukin-12 (IL-12) secretion. L. major infection is associated with self-limiting cutaneous disease and powerful immunity. In stark contrast, the causative agent of visceral leishmaniasis, Leishmania donovani, does not prime human DC for IL-12 production. Here, we report that DC priming by L. major infection results in the early activation of NF-κB transcription factors and the up-regulation and nuclear translocation of interferon regulatory factor 1 (IRF-1) and IRF-8. The inhibition of NF-κB activation by the pretreatment of DC with caffeic acid phenethyl ester blocks L. major-induced IRF-1 and IRF-8 activation and IL-12 expression. We further demonstrate that IRF-1 and IRF-8 obtained from L. major-infected human DC specifically bind to their consensus binding sites on the IL-12p35 promoter, indicating that L. major infection either directly stimulates a signaling cascade or induces an autocrine pathway that activates IRF-1 and IRF-8, ultimately resulting in IL-12 transcription. PMID:18316378

Granule Exocytosis Contributes to Priming and Activation of the Human Neutrophil Respiratory Burst

PubMed Central

Uriarte, Silvia M.; Rane, Madhavi J.; Luerman, Gregory C.; Barati, Michelle T.; Ward, Richard A.; Nauseef, William M.; McLeish, Kenneth R.

2013-01-01

The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT–SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT–SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT–SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT–SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT–SNAP-23 inhibited the increase in plasma membrane expression of gp91phox in TNF-α–primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase. PMID:21642540

A Positive Feedback Loop between Glial Cells Missing 1 and Human Chorionic Gonadotropin (hCG) Regulates Placental hCGβ Expression and Cell Differentiation

PubMed Central

Cheong, Mei-Leng; Wang, Liang-Jie; Chuang, Pei-Yun; Chang, Ching-Wen; Lee, Yun-Shien; Lo, Hsiao-Fan; Tsai, Ming-Song

2015-01-01

Human chorionic gonadotropin (hCG) is composed of a common α subunit and a placenta-specific β subunit. Importantly, hCG is highly expressed in the differentiated and multinucleated syncytiotrophoblast, which is formed via trophoblast cell fusion and stimulated by cyclic AMP (cAMP). Although the ubiquitous activating protein 2 (AP2) transcription factors TFAP2A and TFAP2C may regulate hCGβ expression, it remains unclear how cAMP stimulates placenta-specific hCGβ gene expression and trophoblastic differentiation. Here we demonstrated that the placental transcription factor glial cells missing 1 (GCM1) binds to a highly conserved promoter region in all six hCGβ paralogues by chromatin immunoprecipitation-on-chip (ChIP-chip) analyses. We further showed that cAMP stimulates GCM1 and the CBP coactivator to activate the hCGβ promoter through a GCM1-binding site (GBS1), which also constitutes a previously identified AP2 site. Given that TFAP2C may compete with GCM1 for GBS1, cAMP enhances the association between the hCGβ promoter and GCM1 but not TFAP2C. Indeed, the hCG-cAMP-protein kinase A (PKA) signaling pathway also stimulates Ser269 and Ser275 phosphorylation of GCM1, which recruits CBP to mediate GCM1 acetylation and stabilization. Consequently, hCG stimulates the expression of GCM1 target genes, including the fusogenic protein syncytin-1, to promote placental cell fusion. Our study reveals a positive feedback loop between GCM1 and hCG regulating placental hCGβ expression and cell differentiation. PMID:26503785

Modification of tissue-factor mRNA and protein response to thrombin and interleukin 1 by high glucose in cultured human endothelial cells.

PubMed

Boeri, D; Almus, F E; Maiello, M; Cagliero, E; Rao, L V; Lorenzi, M

1989-02-01

Because diabetic vascular disease is accompanied by a state of hypercoagulability, manifested by increased thrombin activity and foci of intravascular coagulation, we investigated whether a specific procoagulant property of the endothelium--production and surface expression of tissue factor--is modified by elevated glucose concentrations. In unperturbed human vascular endothelial cells, tissue factor mRNA and expression of the functional protein were undetectable and were not induced by 10-12 days of exposure to 30 mM glucose. In thrombin-stimulated cultures, tissue-factor expression was related inversely to cellular density, with confluent cultures producing (per 10(5) cells) half the amount of tissue factor measured in sparse cultures. Cells exposed to high glucose and studied when cell number and thymidine incorporation were identical to control cells manifested increased tissue-factor mRNA level and functional protein production in response to thrombin (P = .002). This effect was not attributable to hypertonicity and was not observed after short exposure to high glucose. In contrast, the tissue-factor response to interleukin 1, a modulator of endothelial function in the context of host defense, was decreased in cells cultured in high glucose (P = .04). These findings indicate that exposure to high glucose can alter tissue-factor gene expression in perturbed vascular endothelium. The reciprocal effects of high glucose on the tissue-factor response to thrombin and interleukin 1 points to different pathways of tissue-factor stimulation by the two agents and suggests functional consequences pertinent to the increased thrombin activity and compromised host-defense mechanisms observed in diabetes.

Cellular myeloperoxidase activity in human monocytes stimulated by hyposialylated immunoglobulins and rheumatoid factors.

PubMed Central

Dodon, M D; Gazzolo, L; Quash, G A

1984-01-01

When hyposialylated , immunoglobulins become immunogenic and tend to form aggregates. In pursuit of the possibility that hyposialylated immunoglobulins (hs-Ig) can trigger human mononuclear phagocytic cells, we have investigated the effects of such hs-Ig on the myeloperoxidase (MPO) activity of these cells. The incubation of human monocytes with aggregated hs-Ig leads to the decrease of intracellular MPO activity. This decrease is dependent on the incubation time, on the amount of hs-Ig added, and on the degree of aggregation. Incubation with unaggregated hs-Ig has a similar effect, thus providing evidence that the loss of sialic acid residues per se is enough to render these molecules capable of decreasing the MPO content of phagocytic cells. Furthermore, human rheumatoid factors, isolated from the sera of rheumatoid arthritis patients, and previously characterized as hyposailylated Ig, interact in the same way with monocytes in triggering the MPO decrease. These observations imply that hs-Ig may be considered as active stimuli in the induction of inflammatory processes, through the initiation of oxidative reactions. PMID:6329948

Regulation of murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in Chinese hamster ovary cells

PubMed Central

Lansdell, K A; Kidd, J F; Delaney, S J; Wainwright, B J; Sheppard, D N

1998-01-01

We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside-out configuration of the patch-clamp technique.The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP-stimulated iodide efflux. However, PKC did not augment the single-channel activity of either human or murine CFTR Cl− channels that had previously been activated by protein kinase A.Fluoride, a non-specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl− channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP-stimulated iodide efflux.The alkaline phosphatase inhibitor, (−)-bromotetramisole augmented cAMP-stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)-bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single-channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR.As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single-channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR. PMID:9769419

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

PubMed

Dwyer, Amy R; Mouchemore, Kellie A; Steer, James H; Sunderland, Andrew J; Sampaio, Natalia G; Greenland, Eloise L; Joyce, David A; Pixley, Fiona J

2016-07-01

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1. © Society for Leukocyte Biology.

Supernatants from oral epithelial cells and gingival fibroblasts modulate human immunodeficiency virus type 1 promoter activation induced by periodontopathogens in monocytes/macrophages.

PubMed

González, O A; Ebersole, J L; Huang, C B

2010-04-01

Bacterial and host cell products during coinfections of Human Immunodeficiency Virus type 1-positive (HIV-1(+)) patients regulate HIV-1 recrudescence in latently infected cells (e.g. T cells, monocytes/macrophages), impacting highly active antiretroviral therapy (HAART) failure and progression of acquired immunodeficiency syndrome. A high frequency of oral opportunistic infections (e.g. periodontitis) in HIV-1(+) patients has been demonstrated; however, their potential to impact HIV-1 exacerbation is unclear. We sought to determine the ability of supernatants derived from oral epithelial cells (OKF4) and human gingival fibroblasts (Gin-4) challenged with periodontal pathogens, to modulate the HIV-1 promoter activation in monocytes/macrophages. BF24 monocytes/macrophages transfected with the HIV-1 promoter driving the expression of chloramphenicol acetyltransferase (CAT) were stimulated with Porphyromonas gingivalis, Fusobacterium nucleatum, or Treponema denticola in the presence of supernatants from OKF4 or Gin4 cells either unstimulated or previously pulsed with bacteria. CAT levels were determined by enzyme-linked immunosorbent assay and cytokine production was evaluated by Luminex beadlyte assays. OKF4 and Gin4 supernatants enhanced HIV-1 promoter activation particularly related to F. nucleatum challenge. An additive effect was observed in HIV-1 promoter activation when monocytes/macrophages were simultaneously stimulated with gingival cell supernatants and bacterial extracts. OKF4 cells produced higher levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukins -6 and -8 in response to F. nucleatum and P. gingivalis. Preincubation of OKF4 supernatants with anti-GM-CSF reduced the additive effect in periodontopathogen-induced HIV-1 promoter activation. These results suggest that soluble mediators produced by gingival resident cells in response to periodontopathogens could contribute to HIV-1 promoter activation in monocytes/macrophages, albeit this effect is most notable following direct stimulation of the cells with oral gram-negative bacteria.

Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larsen, Anett K.; Department of Pharmacology, Institute of Medical Biology, University of Tromso, Tromso; Seternes, Ole-Morten

2008-08-01

In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-{kappa}B) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-inducedmore » cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-{kappa}B-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts.« less

[Study of the antitumor activity of alveolar macrophages after transfected human INF-γ gene].

PubMed

Zhou, Fengli; Bi, Xiaogang; Zhang, Tiantuo; Huang, Jing

2011-05-01

Alveolar macrophages (AMs) activated have the antitumor activity. The interferon-γ (INF-γ) is one of the stimulating factors. INF-γ can enhance the immune function of AMs in vitro. The aim of this study is to investigate the effect of human INF-γ gene on the antitumor activity of AMs when transfected into the alveolar macrophages (AMs) from the patients with lung cancer in vitro. AMs obtained by brochoalveolar lavage were separated and transfected by INF-γ gene. RT-PCR and ELISA were applied to determine whether the transfection was successful. The levels of tumor necrosis factor α (TNF-α), nitric oxide (NO) and interleukin-1 (IL-1) produced by AMs and the killing activity of AMs against L1210 cells was detected respectively. Both RT-PCR and ELISA demonstrated that human INF-γ gene had been successfully transfected into AMs. When transfected by human INF-γ gene, the levels of TNF-α, NO and IL-1 produced by AMs from the patients with lung cancer and the killing activity of AMs against L1210 cells were significantly higher than those of the control groups. Human INF-γ gene can enhance the antitumor activity of AMs when transfected into AMs from the patients with lung cancer.

Anti-melanogenic effects of resveratryl triglycolate, a novel hybrid compound derived by esterification of resveratrol with glycolic acid.

PubMed

Park, Soojin; Seok, Jin Kyung; Kwak, Jun Yup; Choi, Yun-Hyeok; Hong, Seong Su; Suh, Hwa-Jin; Park, Woncheol; Boo, Yong Chool

2016-07-01

Resveratrol is known to inhibit cellular melanin synthesis by multiple mechanisms. Glycolic acid (GA) is used in skin care products for its excellent skin penetration. The purpose of this study was to examine the anti-melanogenic effects of resveratryl triglycolate (RTG), a novel hybrid compound of resveratrol and GA, in comparison with resveratrol, GA, resveratryl triacetate (RTA) and arbutin. Resveratrol, RTG, and RTA inhibited the catalytic activity human tyrosinase (TYR) more potently than arbutin or GA did. Their cytotoxic and anti-melanogenic effects were examined using murine melanoma B16/F10 cells and human epidermal melanocytes (HEMs). The cytotoxicity of RTG was similar to that of resveratrol and RTA. RTG at 3-10 μM decreased melanin levels and cellular TYR activities in α-melanocyte-stimulating hormone-stimulated B16/F10 cells, and L-tyrosine-stimulated HEMs. RTG also suppressed mRNA and protein expression of TYR, tyrosinase-related protein 1, L-3,4-dihydroxyphenylalanine chrome tautomerase, and microphthalmia-associated transcription factor (MITF) in HEMs stimulated with L-tyrosine. This study suggests that, like resveratrol and RTA, RTG can attenuate cellular melanin synthesis effectively through the suppression of MITF-dependent expression of melanogenic enzymes and the inhibition of catalytic activity of TYR enzyme. RTG therefore has potential for use as a cosmeceutical ingredient for skin whitening.

T cell-replacing factor for glucocorticosteroid-induced immunoglobulin production. A unique steroid-dependent cytokine

PubMed Central

1983-01-01

Glucocorticosteroids (GCS) added to otherwise unstimulated cultures of human peripheral blood mononuclear cells (PBMC) induce the synthesis and secretion of all classes of immunoglobulin. The magnitude of this response is similar to that seen with other polyclonal B cell activators such as pokeweed mitogen (PWM), and like that of PWM, the steroid effect is dependent on both T cells and monocytes. To determine the cellular target for GCS in these cultures, separated populations of T cells and non-T cells were preincubated with steroids and then recombined. No immunoglobulin was produced in any of these preincubation experiments. As a different approach to this question, supernatants were collected from various cell populations following stimulation with PWM, concanavalin A (Con A), phytohemagglutinin (PHA), alloantigens, or GCS. These supernatants were tested for their effects on GCS-induced Ig production by B cells. Supernatants from 3-d cultures of unstimulated, as well as GCS-treated, PBMC contained a T cell- replacing factor that permitted T-depleted PBMC to produce Ig upon steroid stimulation. This supernatant factor (TRF-S) could be produced in the absence of steroid stimulation, but both the factor and GCS were necessary for the induction of Ig synthesis. Production of the TRF-S required the presence of both T cells and adherent cells in culture and was found in the highest concentrations at 3-4 d of culture. Supernatants from cultures stimulated with PWM, PHA, Con A, and alloantigens did not contain detectable TRF-S activity, and TRF-S was unable to replace helper T cells for PWM-induced Ig production. TRF-S required the presence of adherent cells in the T cell-depleted responder population for its action. Further, it was effective in inducing Ig production along with GCS in the presence of a sufficient concentration of cyclosporin A to block all T cell helper activity for primary responses of PBMC to PWM or GCS. TRF-S was inactivated by trypsin treatment, heating to 56 degrees C, freezing, lyophilization, and storage at 4 degrees C for greater than 3 wk. Its molecular weight is probably 10,000 daltons or more, since TRF-S activity is not rapidly dialyzable. These experiments indicate that GCS-induced Ig production by human B cells does not require the presence of intact T cells in the cultures and therefore the steroids are not exerting their influence directly on T suppressor or T helper cells. Furthermore, they demonstrate a previously unrecognized cytokine that induces the differentiation of human B cells to Ig production in the presence of GCS. PMID:6605406

Heparin-binding growth factor isolated from human prostatic extracts.

PubMed

Mydlo, J H; Bulbul, M A; Richon, V M; Heston, W D; Fair, W R

1988-01-01

Prostatic tissue extracts from patients with benign prostatic hyperplasia (BPH) and prostatic carcinoma were fractionated using heparin-Sepharose chromatography. The mitogenic activity of eluted fractions on quiescent subconfluent Swiss Albino 3T3 fibroblasts was tested employing a tritiated-thymidine-incorporation assay. Two peaks of activity were consistently noted--one in the void volume and a second fraction which eluted with 1.3-1.6 M NaCl and contained the majority of the mitogenic activity. Both non-heparin- and heparin-binding fractions increased tritiated incorporation into a mouse osteoblast cell line (MC3T3), while only the heparin-binding fractions stimulated a human umbilical vein endothelial cell line (HUV). No increased uptake of thymidine was seen using a human prostatic carcinoma cell line (PC-3). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of lyophilized active fractions showed a persistent band at 17,500 daltons. The purified protein demonstrated angiogenic properties using the chick embryo chorioallantoic membrane (CAM) assay. Western blot analysis using antibodies specific to basic fibroblast growth factor (bFGF) or acidic FGF (aFGF) demonstrated that the former, but not the latter, bound to prostatic growth factor (PrGF), and inhibited its mitogenic activity as well. It appears that PrGF shares homology with basic fibroblast growth factors.

Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism.

PubMed

Lee, Young Ah; Nam, Young Hee; Min, Arim; Kim, Kyeong Ah; Nozaki, Tomoyoshi; Saito-Nakano, Yumiko; Mirelman, David; Shin, Myeong Heon

2014-01-01

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. © Y.A. Lee et al., published by EDP Sciences, 2014.

Identification of a novel cyclosporin-sensitive element in the human tumor necrosis factor alpha gene promoter

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha), a cytokine with pleiotropic biological effects, is produced by a variety of cell types in response to induction by diverse stimuli. In this paper, TNF-alpha mRNA is shown to be highly induced in a murine T cell clone by stimulation with T cell receptor (TCR) ligands or by calcium ionophores alone. Induction is rapid, does not require de novo protein synthesis, and is completely blocked by the immunosuppressant cyclosporin A (CsA). We have identified a human TNF-alpha promoter element, kappa 3, which plays a key role in the calcium-mediated inducibility and CsA sensitivity of the gene. In electrophoretic mobility shift assays, an oligonucleotide containing kappa 3 forms two DNA protein complexes with proteins that are present in extracts from unstimulated T cells. These complexes appear in nuclear extracts only after T cell stimulation. Induction of the inducible nuclear complexes is rapid, independent of protein synthesis, and blocked by CsA, and thus, exactly parallels the induction of TNF-alpha mRNA by TCR ligands or by calcium ionophore. Our studies indicate that the kappa 3 binding factor resembles the preexisting component of nuclear factor of activated T cells. Thus, the TNF-alpha gene is an immediate early gene in activated T cells and provides a new model system in which to study CsA-sensitive gene induction in activated T cells. PMID:8376940

Noninvasive near-infrared topography of human brain activity using intensity modulation spectroscopy

NASA Astrophysics Data System (ADS)

Yamashita, Yuichi; Maki, Atsushi; Ito, Yoshitoshi; Watanabe, Eiju; Mayanagi, Yoshiaki; Koizumi, Hideaki

1996-04-01

We describe the functional topography of human brain activity due to motor stimulation by using near-infrared spectroscopy. Finger motion by each hand was used as the motor stimulation, and activity in the left fronto-central region of the brain was measured. A greater change in oxyhemoglobin concentration due to brain activity during the stimulation was obtained for the right hand than for the left hand. Localization of the activity was obtained by topographically mapping the measured changes for ten positions within the region.

Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals.

PubMed

Edmead, C E; Patel, Y I; Wilson, A; Boulougouris, G; Hall, N D; Ward, S G; Sansom, D M

1996-10-15

A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways.

Early innate immune responses to Sin Nombre hantavirus occur independently of IFN regulatory factor 3, characterized pattern recognition receptors, and viral entry.

PubMed

Prescott, Joseph B; Hall, Pamela R; Bondu-Hawkins, Virginie S; Ye, Chunyan; Hjelle, Brian

2007-08-01

Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.

Cryopreservation induces macrophage colony stimulating factor from human periodontal ligament cells in vitro.

PubMed

Rhim, E-M; Ahn, S-J; Kim, J-Y; Chang, Y-R; Kim, K-H; Lee, H-W; Jung, S-H; Kim, E-C; Park, S-H

2013-10-01

Cryopreservation is used to protect vital periodontal ligaments during the transplantation of teeth. We investigated which gene products implicated in root resorption are upregulated in human periodontal ligament cells by cryopreservation, and whether cryopreservation affects the expression of macrophage-colony stimulating factor (M-CSF) in human periodontal ligament cells. We used customized microarrays to compare gene expression in human periodontal ligament cells cultured from teeth immediately after extraction and from cryopreserved teeth. Based on the result of these assays, we examined M-CSF expression in periodontal ligament cells from the immediately extracted tooth and cryopreserved teeth by real-time PCR, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence. We also investigated whether human bone marrow cells differentiate into tartrate-resistant acid phosphatase (TRAP) positive osteoclasts when stimulated with RANKL (Receptor Activator for Nuclear Factor κ B Ligand) together with any secreted M-CSF present in the supernatants of the periodontal ligament cells cultured from the various groups of teeth. M-CSF was twofold higher in the periodontal ligament cells from the rapid freezing teeth than in those from the immediately extracted group (p < 0.05). Cryopreservation increased M-CSF expression in the periodontal ligament cells when analyzed by real time PCR, ELISA, Western blotting, and immunofluorescence (p < 0.05). TRAP positive osteoclasts were formed in response to RANKL and the secreted M-CSF present in the supernatants of all the experimental groups except negative control. These results demonstrate that cryopreservation promotes the production of M-CSF, which plays an important role in root resorption by periodontal ligament cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Compound C Stimulates Heme Oxygenase-1 Gene Expression via the Nrf2-ARE Pathway to Preserve Human Endothelial Cell Survival

PubMed Central

Liu, Xiao-ming; Peyton, Kelly J.; Shebib, Ahmad R.; Wang, Hong; Durante, William

2011-01-01

We recently identified adenosine monophosphate-activated protein kinase (AMPK) as a novel inducer of heme oxygenase-1 (HO-1) and surprisingly found that compound C (6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]3-pyridin-4-yl-pyrazolo[1,5-a] pyrimidine), a cell-permeable inhibitor of AMPK, could also elevate HO-1 suggesting other AMPK-independent actions for this agent. In this study, we investigated the biochemical mechanism by which compound C stimulates HO-1 expression in human endothelial cells (ECs) and determined the biological significance of the induction of HO-1 by compound C in these cells. Compound C stimulated a concentration- and time-dependent increase in HO-1 expression and an increase in HO-1 promoter activity that was abrogated by mutating the antioxidant responsive elements (AREs) in the HO-1 promoter or by overexpressing a dominant negative mutant of NF-E2-related factor-2 (Nrf2). Compound C also stimulated Nrf2 expression and this was associated with an increase in the production of reactive oxygen species and with a decline in intracellular glutathione levels. Interestingly, the glutathione donor N-acetyl-L-cysteine or the NADPH oxidase inhibitor apocynin blocked the induction of HO-1 by compound C. Finally, compound C stimulated EC death and this was potentiated by silencing HO-1 expression and reversed by the administration of CO, biliverdin, or bilirubin. In conclusion, this study demonstrates that compound C stimulates HO-1 gene expression in human vascular endothelium via the activation of the Nrf2/ARE signaling pathway to counteract compound C-mediated cell death. The ability of compound C to induce HO-1 expression may contribute to the pleiotropic actions of this agent and suggest caution when using compound C to probe for AMPK functions. PMID:21635873

Apolipoprotein CIII-induced THP-1 cell adhesion to endothelial cells involves pertussis toxin-sensitive G protein- and protein kinase C alpha-mediated nuclear factor-kappaB activation.

PubMed

Kawakami, Akio; Aikawa, Masanori; Nitta, Noriko; Yoshida, Masayuki; Libby, Peter; Sacks, Frank M

2007-01-01

Plasma apolipoprotein CIII (apoCIII) independently predicts risk for coronary heart disease (CHD). We recently reported that apoCIII directly enhances adhesion of human monocytes to endothelial cells (ECs), and identified the activation of PKC alpha as a necessary upstream event of enhanced monocyte adhesion. This study tested the hypothesis that apoCIII activates PKC alpha in human monocytic THP-1 cells, leading to NF-kappaB activation. Among inhibitors specific to PKC activators, phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609 limited apoCIII-induced PKC alpha activation and THP-1 cell adhesion. ApoCIII increased PC-PLC activity in THP-1 cells, resulting in PKC alpha activation. Pertussis toxin (PTX) inhibited apoCIII-induced PC-PLC activation and subsequent PKC alpha activation, implicating PTX-sensitive G protein pathway. ApoCIII further activated nuclear factor-kappaB (NF-kappaB) through PKC alpha in THP-1 cells and augmented beta1-integrin expression. The NF-kappaB inhibitor peptide SN50 partially inhibited apoCIII-induced beta1-integrin expression and THP-1 cell adhesion. ApoCIII-rich VLDL had similar effects to apoCIII alone. PTX-sensitive G protein pathway participates critically in PKC alpha stimulation in THP-1 cells exposed to apoCIII, activating NF-kappaB, and increasing beta1-integrin. This action causes monocytic cells to adhere to endothelial cells. Furthermore, because leukocyte NF-kappaB activation contributes to inflammatory aspects of atherogenesis, apoCIII may stimulate diverse inflammatory responses through monocyte activation.

Cyclic hydrostatic pressure and particles increase synthesis of 1,25-dihydroxyvitamin D3 by human macrophages in vitro.

PubMed

Evans, C E; Mylchreest, S; Mee, A P; Berry, J L; Andrew, J G

2006-01-01

1,25-Dihydroxyvitamin D(3) has a pivotal role in bone resorption and osteoclast activity. As activated macrophages are known to synthesise 1,25-dihydroxyvitamin D(3), this study examined whether pressure modulated its synthesis. Pressure and particles have been shown to increase synthesis of pro-resorptive cytokines and other factors by cultured macrophages. Human peripheral blood macrophages were isolated, cultured and exposed to pressure (similar to that found in the human joint) and/or particles. Synthesis of 1,25-dihydroxyvitamin D(3) by macrophages was assayed using high pressure liquid chromatography and in situ hybridization. Synthesis of 1,25-dihydroxyvitamin D(3) but not 24,25-dihydroxyvitamin D(3) was increased in macrophages under pressure. In situ hybridization demonstrated an increase in 1alpha-hydroxylase expression in response to pressure or particles and simultaneous exposure to both stimuli generated higher expression of 1alpha-hydroxylase. In conclusion, this is the first study to demonstrate that mechanical loading, in the form of pressure, stimulates 1,25-dihydroxyvitamin D(3) synthesis in human macrophages. These findings have implications for the in vivo situation, as they suggest that 1,25-dihydroxyvitamin D(3) could be one factor stimulating osteoclastic bone resorption in pathologies, such as arthritis or implant loosening, where intra-articular or intra-osseous pressure is raised or where wear particles interact with macrophages.

Beta-glucan-depleted, glycopeptide-rich extracts from Brewer's and Baker's yeast (Saccharomyces cerevisiae) lower interferon-gamma production by stimulated human blood cells in vitro.

PubMed

Williams, Roderick; Dias, Daniel A; Jayasinghe, Nirupama; Roessner, Ute; Bennett, Louise E

2016-04-15

Regulation of the human immune system requires controlled pro- and anti-inflammatory responses for host defence against infection and disease states. Yeasts (Saccharomyces cerevisiae), as used in brewing and baking, are mostly known for ability to stimulate the human immune-system predominantly reflecting the pro-inflammatory cell wall β-glucans. However, in this study, using food-compatible processing methods, glycopeptide-enriched and β-glucan-depleted products were each prepared from Brewer's and Baker's yeasts, which suppressed production of interferon-γ (IFN-γ) in human whole blood cell assay, signifying that anti-inflammatory factors are also present in yeast. Anti-inflammatory bioactivities of products prepared from Brewer's and Baker's yeast were compared with the commercial yeast product, Epicor®. While unfractionated Epicor was inactive, the C18 resin-binding fractions of Brewer's and Baker's yeast products and Epicor dose-dependently lowered IFN-γ, demonstrating that Epicor also contained both pro-inflammatory (β-glucans) and anti-inflammatory components. Anti-inflammatory activity was attributed to C18 resin-binding species glyco-peptides in Epicor and experimental yeast products. This study demonstrated that pro- and anti-inflammatory factors could be resolved and enriched in yeasts by suitable processing, with potential to improve specific activities. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Local entrainment of oscillatory activity induced by direct brain stimulation in humans

PubMed Central

Amengual, Julià L.; Vernet, Marine; Adam, Claude; Valero-Cabré, Antoni

2017-01-01

In a quest for direct evidence of oscillation entrainment, we analyzed intracerebral electroencephalographic recordings obtained during intracranial electrical stimulation in a cohort of three medication-resistant epilepsy patients tested pre-surgically. Spectral analyses of non-epileptogenic cerebral sites stimulated directly with high frequency electrical bursts yielded episodic local enhancements of frequency-specific rhythmic activity, phase-locked to each individual pulse. These outcomes reveal an entrainment of physiological oscillatory activity within a frequency band dictated by the rhythm of the stimulation source. Our results support future uses of rhythmic stimulation to elucidate the causal contributions of synchrony to specific aspects of human cognition and to further develop the therapeutic manipulation of dysfunctional rhythmic activity subtending the symptoms of some neuropsychiatric conditions. PMID:28256510

Role of the autonomic nervous system in activation of human brown adipose tissue: A review of the literature.

PubMed

Bahler, L; Molenaars, R J; Verberne, H J; Holleman, F

2015-12-01

Brown adipose tissue (BAT) is able to convert calories into heat rather than storing them. Therefore, activated BAT could be a potential target in the battle against obesity and type 2 diabetes. This review focuses on the role of the autonomic nervous system in the activation of human BAT. Although the number of studies focusing on BAT in humans is limited, involvement of the sympathetic nervous system (SNS) in BAT activation is evident. Metabolic BAT activity can be visualized with (18)F-fluorodeoxyglucose, whereas sympathetic activation of BAT can be visualized with nuclear-medicine techniques using different radiopharmaceuticals. Also, interruption of the sympathetic nerves leading to BAT activation diminishes sympathetic stimulation, resulting in reduced metabolic BAT activity. Furthermore, both β- and α-adrenoceptors might be important in the stimulation process of BAT, as pretreatment with propranolol or α-adrenoceptor blockade also diminishes BAT activity. In contrast, high catecholamine levels are known to activate and recruit BAT. There are several interventional studies in which BAT was successfully inhibited, whereas only one interventional study aiming to activate BAT resulted in the intended outcome. Most studies have focused on the SNS for activating BAT, although the parasympathetic nervous system might also be a target of interest. To better define the possible role of BAT in strategies to combat the obesity epidemic, it seems likely that future studies focusing on both histology and imaging are essential for identifying the factors and receptors critical for activation of human BAT. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding

PubMed Central

Hymowitz, Sarah G.; Filvaroff, Ellen H.; Yin, JianPing; Lee, James; Cai, Liping; Risser, Philip; Maruoka, Miko; Mao, Weiguang; Foster, Jessica; Kelley, Robert F.; Pan, Guohua; Gurney, Austin L.; de Vos, Abraham M.; Starovasnik, Melissa A.

2001-01-01

The proinflammatory cytokine interleukin 17 (IL-17) is the founding member of a family of secreted proteins that elicit potent cellular responses. We report a novel human IL-17 homolog, IL-17F, and show that it is expressed by activated T cells, can stimulate production of other cytokines such as IL-6, IL-8 and granulocyte colony-stimulating factor, and can regulate cartilage matrix turnover. Unexpectedly, the crystal structure of IL-17F reveals that IL-17 family members adopt a monomer fold typical of cystine knot growth factors, despite lacking the disulfide responsible for defining the canonical ‘knot’ structure. IL-17F dimerizes in a parallel manner like neurotrophins, and features an unusually large cavity on its surface. Remarkably, this cavity is located in precisely the same position where nerve growth factor binds its high affinity receptor, TrkA, suggesting further parallels between IL-17s and neurotrophins with respect to receptor recognition. PMID:11574464

Suppression of in vitro murine T cell proliferation by human adipose tissue-derived mesenchymal stem cells is dependent mainly on cyclooxygenase-2 expression

PubMed Central

Kim, Jin-Hee; Lee, Yong-Taek; Hong, Jun Man

2013-01-01

Mesenchymal stem cells (MSCs) of human origin have been frequently applied to experimental animal models to evaluate their immunomodulatory functions. MSCs are known to be activated by cytokines from T cells, predominantly by interferon-γ (IFN-γ), in conjunction with other cytokines such as tumor necrosis factor-α (TNF-α) and interlukin-1β. Because IFN-γ is not cross-reactive between human and mouse species, the manner in which human MSCs administered in experimental animals are activated and stimulated to function has been questioned. In the present study, we established MSCs from human adipose tissue. They successfully suppressed the proliferation of not only human peripheral blood mononuclear cells but also mouse splenic T cells. When these human MSCs were stimulated with a culture supernatant of mouse T cells or recombinant murine TNF-α, they expressed cyclooxygenase-2 (COX-2), but not indoleamine 2,3-dioxygenase. The dominant role of COX-2 in suppressing mouse T cell proliferation was validated by the addition of COX-2 inhibitor in the co-culture, wherein the suppressed proliferation was almost completely recovered. In conclusion, human MSCs in a murine environment were activated, at least in part, by TNF-α and mainly used COX-2 as a tool for the suppression of in vitro T cell proliferation. These results should be considered when interpreting results for human MSCs in experimental animals. PMID:24386599

Effects of extracellular magnesium on the differentiation and function of human osteoclasts.

PubMed

Wu, Lili; Luthringer, Bérengère J C; Feyerabend, Frank; Schilling, Arndt F; Willumeit, Regine

2014-06-01

Magnesium-based implants have been shown to influence the surrounding bone structure. In an attempt to partially reveal the cellular mechanisms involved in the remodelling of magnesium-based implants, the influence of increased extracellular magnesium content on human osteoclasts was studied. Peripheral blood mononuclear cells were driven towards an osteoclastogenesis pathway via stimulation with receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor for 28 days. Concomitantly, the cultures were exposed to variable magnesium concentrations (from either magnesium chloride or magnesium extracts). Osteoclast proliferation and differentiation were evaluated based on cell metabolic activity, total protein content, tartrate-resistant acid phosphatase activity, cathepsin K and calcitonin receptor immunocytochemistry, and cellular ability to form resorption pits. While magnesium chloride first enhanced and then opposed cell proliferation and differentiation in a concentration-dependent manner (peaking between 10 and 15mM magnesium chloride), magnesium extracts (with lower magnesium contents) appeared to decrease cell metabolic activity (≈50% decrease at day 28) while increasing osteoclast activity at a lower concentration (twofold higher). Together, the results indicated that (i) variations in the in vitro extracellular magnesium concentration affect osteoclast metabolism and (ii) magnesium extracts should be used preferentially in vitro to more closely mimic the in vivo environment. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Kinetics of GLUT4 Trafficking in Rat and Human Skeletal Muscle

PubMed Central

Karlsson, Håkan K.R.; Chibalin, Alexander V.; Koistinen, Heikki A.; Yang, Jing; Koumanov, Francoise; Wallberg-Henriksson, Harriet; Zierath, Juleen R.; Holman, Geoffrey D.

2009-01-01

OBJECTIVE In skeletal muscle, insulin stimulates glucose transport activity three- to fourfold, and a large part of this stimulation is associated with a net translocation of GLUT4 from an intracellular compartment to the cell surface. We examined the extent to which insulin or the AMP-activated protein kinase activator AICAR can lead to a stimulation of the exocytosis limb of the GLUT4 translocation pathway and thereby account for the net increase in glucose transport activity. RESEARCH DESIGN AND METHODS Using a biotinylated photoaffinity label, we tagged endogenous GLUT4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or AICAR. Isolated epitrochlearis muscles were obtained from male Wistar rats. Vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men (age 39 ± 11 years and BMI 25.8 ± 0.8 kg/m2). RESULTS In rat epitrochlearis muscle, insulin exposure leads to a sixfold stimulation of the GLUT4 exocytosis rate (with basal and insulin-stimulated rate constants of 0.010 and 0.067 min−1, respectively). In human vastus lateralis muscle, insulin stimulates GLUT4 translocation by a similar sixfold increase in the exocytosis rate constant (with basal and insulin-stimulated rate constants of 0.011 and 0.075 min−1, respectively). In contrast, AICAR treatment does not markedly increase exocytosis in either rat or human muscle. CONCLUSIONS Insulin stimulation of the GLUT4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle. PMID:19188436

Innate defense regulator IDR-1018 activates human mast cells through G protein-, phospholipase C-, MAPK- and NF-ĸB-sensitive pathways.

PubMed

Yanashima, Kensuke; Chieosilapatham, Panjit; Yoshimoto, Eri; Okumura, Ko; Ogawa, Hideoki; Niyonsaba, François

2017-08-01

Host defense (antimicrobial) peptides not only display antimicrobial activities against numerous pathogens but also exert a broader spectrum of immune-modulating functions. Innate defense regulators (IDRs) are a class of host defense peptides synthetically developed from natural or endogenous cationic host defense peptides. Of the IDRs developed to date, IDR-1018 is more efficient not only in killing bacteria but also in regulating the various functions of macrophages and neutrophils and accelerating the wound healing process. Because mast cells intimately participate in wound healing and a number of host defense peptides involved in wound healing are also known to activate mast cells, this study aimed to investigate the effects of IDR-1018 on mast cell activation. Here, we showed that IDR-1018 induced the degranulation of LAD2 human mast cells and caused their production of leukotrienes, prostaglandins and various cytokines and chemokines, including granulocyte-macrophage colony-stimulating factor, interleukin-8, monocyte chemoattractant protein-1 and -3, macrophage-inflammatory protein-1α and -1β, and tumor necrosis factor-α. Furthermore, IDR-1018 increased intracellular calcium mobilization and induced mast cell chemotaxis. The mast cell activation was markedly suppressed by pertussis toxin, U-73122, U0126, SB203580, JNK inhibitor II, and NF-κB activation inhibitor II, suggesting the involvement of G-protein, phospholipase C, ERK, p38, JNK and NF-κB pathways, respectively, in IDR-1018-induced mast cell activation. Notably, we confirmed that IDR-1018 caused the phosphorylation of MAPKs and IκB. Altogether, the current study suggests a novel immunomodulatory role of IDR-1018 through its ability to recruit and activate human mast cells at the sites of inflammation and wounds. We report that IDR-1018 stimulates various functions of human mast cells. IDR-1018-induced mast cell activation is mediated through G protein, PLC, MAPK and NF-κB pathways. IDR-1018 will be a useful therapeutic agent for wound healing.

Cell-line-specific stimulation of tumor cell aggressiveness by wound healing factors - a central role for STAT3.

PubMed

Ekblad, Lars; Lindgren, Gustaf; Persson, Emma; Kjellén, Elisabeth; Wennerberg, Johan

2013-01-25

Local recurrence is a major factor affecting survival after treatment for head and neck squamous cell carcinoma (HNSCC). It is possible that the normal processes involved in wound healing after surgical removal of a primary tumor can boost the regrowth of residual cancer cells, thereby contributing to the recurrent growth. In this work, we collected human wound fluids and used them to investigate the effect of wound healing factors on HNSCC cell lines in vitro. Wound fluids were collected from thyroidectomized patients diagnosed with benign disease and were included in assays of cell proliferation, migration, cell scattering, and invasion. The involvement of intracellular signaling pathways and membrane receptors were investigated by western blotting and the inclusion of specific inhibitors. One out of four cell lines was greatly stimulated in proliferation, migration, cell scattering, and invasion by the addition of wound fluid as compared with addition of fetal bovine or human serum. These effects were accompanied by a sharp increase in activation of signal transducer and activator of transcription 3 (STAT3). Inhibition of STAT3 activation abolished the wound fluid response, showing that STAT3 plays an important role in the wound healing response. Several of the observed phenotypic changes were epithelial-to-mesenchymal transition (EMT)-like, but the appropriate changes were not seen in any of the EMT markers investigated. The involvement of c-Met or epidermal growth factor receptor family members was excluded, while the interleukin-6 receptor was found to be partly responsible for the activation of STAT3. In conclusion, we found cell-line-specific effects of wound healing factors on HNSCC, setting the stage for therapy development and predictive opportunities.

SIZE FRACTIONS OF AMBIENT PARTICULATE MATTER INDUCE GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR IN HUMAN BRONCHIAL EPITHELIAL CELLS BY MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS. (R827351C004)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

UV Light Potentiates STING (Stimulator of Interferon Genes)-dependent Innate Immune Signaling through Deregulation of ULK1 (Unc51-like Kinase 1)*

PubMed Central

Kemp, Michael G.; Lindsey-Boltz, Laura A.; Sancar, Aziz

2015-01-01

The mechanism by which ultraviolet (UV) wavelengths of sunlight trigger or exacerbate the symptoms of the autoimmune disorder lupus erythematosus is not known but may involve a role for the innate immune system. Here we show that UV radiation potentiates STING (stimulator of interferon genes)-dependent activation of the immune signaling transcription factor interferon regulatory factor 3 (IRF3) in response to cytosolic DNA and cyclic dinucleotides in keratinocytes and other human cells. Furthermore, we find that modulation of this innate immune response also occurs with UV-mimetic chemical carcinogens and in a manner that is independent of DNA repair and several DNA damage and cell stress response signaling pathways. Rather, we find that the stimulation of STING-dependent IRF3 activation by UV is due to apoptotic signaling-dependent disruption of ULK1 (Unc51-like kinase 1), a pro-autophagic protein that negatively regulates STING. Thus, deregulation of ULK1 signaling by UV-induced DNA damage may contribute to the negative effects of sunlight UV exposure in patients with autoimmune disorders. PMID:25792739

TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

PubMed Central

Posse, Viktor; Shahzad, Saba; Falkenberg, Maria; Hällberg, B. Martin; Gustafsson, Claes M.

2015-01-01

A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery. PMID:25690892

Bacterium induces cryptic meroterpenoid pathway in the pathogenic fungus Aspergillus fumigatus.

PubMed

König, Claudia C; Scherlach, Kirstin; Schroeckh, Volker; Horn, Fabian; Nietzsche, Sandor; Brakhage, Axel A; Hertweck, Christian

2013-05-27

Stimulating encounter: The intimate, physical interaction between the soil-derived bacterium Streptomyces rapamycinicus and the human pathogenic fungus Aspergillus fumigatus led to the activation of an otherwise silent polyketide synthase (PKS) gene cluster coding for an unusual prenylated polyphenol (fumicycline A). The meroterpenoid pathway is regulated by a pathway-specific activator gene as well as by epigenetic factors. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

T-cell receptor signaling enhances transcriptional elongation from latent HIV proviruses by activating P-TEFb through an ERK-dependent pathway.

PubMed

Kim, Young Kyeung; Mbonye, Uri; Hokello, Joseph; Karn, Jonathan

2011-07-29

Latent human immunodeficiency virus (HIV) proviruses are thought to be primarily reactivated in vivo through stimulation of the T-cell receptor (TCR). Activation of the TCR induces multiple signal transduction pathways, leading to the ordered nuclear migration of the HIV transcription initiation factors NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T-cells), as well as potential effects on HIV transcriptional elongation. We have monitored the kinetics of proviral reactivation using chromatin immunoprecipitation assays to measure changes in the distribution of RNA polymerase II in the HIV provirus. Surprisingly, in contrast to TNF-α (tumor necrosis factor α) activation, where early transcription elongation is highly restricted due to rate-limiting concentrations of Tat, efficient and sustained HIV elongation and positive transcription elongation factor b (P-TEFb) recruitment are detected immediately after the activation of latent proviruses through the TCR. Inhibition of NFAT activation by cyclosporine had no effect on either HIV transcription initiation or elongation. However, examination of P-TEFb complexes by gel-filtration chromatography showed that TCR signaling led to the rapid dissociation of the large inactive P-TEFb:7SK RNP (small nuclear RNA 7SK ribonucleoprotein) complex and the release of active low-molecular-weight P-TEFb complexes. Both P-TEFb recruitment to the HIV long terminal repeat and enhanced HIV processivity were blocked by the ERK (extracellular-signal-regulated kinase) inhibitor U0126, but not by AKT (serine/threonine protein kinase Akt) and PI3K (phosphatidylinositol 3-kinase) inhibitors. In contrast to treatment with HMBA (hexamethylene bisacetamide) and DRB (5,6-dichlorobenzimidazole 1-β-ribofuranoside), which disrupt the large 7SK RNP complex but do not stimulate early HIV elongation, TCR signaling provides the first example of a physiological pathway that can shift the balance between the inactive P-TEFb pool and the active P-TEFb pool and thereby stimulate proviral reactivation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Sulforaphane inhibits phorbol ester-stimulated IKK-NF-κB signaling and COX-2 expression in human mammary epithelial cells by targeting NF-κB activating kinase and ERK.

PubMed

Kim, Ha-Na; Kim, Do-Hee; Kim, Eun-Hee; Lee, Mee-Hyun; Kundu, Joydeb Kumar; Na, Hye-Kyung; Cha, Young-Nam; Surh, Young-Joon

2014-08-28

Sulforaphane, an isothiocyanate present in cruciferous vegetables, has been reported to possess anti-inflammatory and cancer chemopreventive properties. However, the molecular mechanisms by which sulforaphane suppresses inflammation and carcinogenesis are yet to be fully elucidated. Since the aberrant expression of cyclooxygenase-2 (COX-2) links inflammation and cancer, the present study was aimed to elucidate the mechanisms by which sulforaphane modulates COX-2 overexpression in human mammary epithelial (MCF-10A) cells stimulated with a prototypic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of MCF-10A cells with sulforaphane significantly inhibited TPA-induced expression of COX-2 protein and its mRNA transcript. Transient transfection of cells with deletion mutant constructs of COX-2 promoter revealed that the transcription factor nuclear factor-kappaB (NF-κB) plays a key role in TPA-induced COX-2 expression in MCF-10A cells. Pretreatment with sulforaphane significantly attenuated nuclear localization, DNA binding and the transcriptional activity of NF-κB through inhibition of phosphorylation and subsequent degradation of IκBα in MCF-10A cells stimulated with TPA. Sulforaphane also attenuated TPA-induced activation of IκB kinases (IKK), NF-κB-activating kinase (NAK) and extracellular signal-regulated kinase-1/2 (ERK1/2). Pharmacological inhibition of IKK or transient transfection of cells with dominant-negative mutant forms of this kinase abrogated TPA-induced NF-κB activation and COX-2 expression. In addition, the blockade of ERK1/2 activation negated the catalytic activity of IKKα, but not that of IKKβ, whereas silencing NAK by specific siRNA abrogated the IKKβ activity in TPA-treated cells. Taken together, sulforaphane inhibits TPA-induced NF-κB activation and COX-2 expression in MCF-10A cells by blocking two distinct signaling pathways mediated by ERK1/2-IKKα and NAK-IKKβ. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Molecular basis of retinol anti-ageing properties in naturally aged human skin in vivo.

PubMed

Shao, Y; He, T; Fisher, G J; Voorhees, J J; Quan, T

2017-02-01

Retinoic acid has been shown to improve the aged-appearing skin. However, less is known about the anti-ageing effects of retinol (ROL, vitamin A), a precursor of retinoic acid, in aged human skin in vivo. This study aimed to investigate the molecular basis of ROL anti-ageing properties in naturally aged human skin in vivo. Sun-protected buttock skin (76 ± 6 years old, n = 12) was topically treated with 0.4% ROL and its vehicle for 7 days. The effects of topical ROL on skin epidermis and dermis were evaluated by immunohistochemistry, in situ hybridization, Northern analysis, real-time RT-PCR and Western analysis. Collagen fibrils nanoscale structure and surface topology were analysed by atomic force microscopy. Topical ROL shows remarkable anti-ageing effects through three major types of skin cells: epidermal keratinocytes, dermal endothelial cells and fibroblasts. Topical ROL significantly increased epidermal thickness by stimulating keratinocytes proliferation and upregulation of c-Jun transcription factor. In addition to epidermal changes, topical ROL significantly improved dermal extracellular matrix (ECM) microenvironment; increasing dermal vascularity by stimulating endothelial cells proliferation and ECM production (type I collagen, fibronectin and elastin) by activating dermal fibroblasts. Topical ROL also stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, and thus enriched the deposition of ECM in aged human skin in vivo. 0.4% topical ROL achieved similar results as seen with topical retinoic acid, the biologically active form of ROL, without causing noticeable signs of retinoid side effects. 0.4% topical ROL shows remarkable anti-ageing effects through improvement of the homeostasis of epidermis and dermis by stimulating the proliferation of keratinocytes and endothelial cells, and activating dermal fibroblasts. These data provide evidence that 0.4% topical ROL is a promising and safe treatment to improve the naturally aged human skin. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

The potent inhibition of vapiprost, a novel thromboxane A2 receptor antagonist, on the secondary aggregation and ATP release of human platelets.

PubMed

Horie, S; Yamada, M; Satoh, M; Noritake, S; Hiraishi, S; Kizaki, K; Kurusu, O; Nakahara, T; Ishii, H; Kazama, M

1997-06-01

The inhibitory effects of vapiprost hydrochloride (vapiprost), a novel thromboxane A2 receptor antagonist, on platelet aggregation and ATP release were studied using platelet rich plasma (PRP) of humans, guinea pigs, rabbits and rats. In in vitro experiments with human platelet, vapiprost inhibited the aggregation and ATP release stimulated with U-46619, collagen or arachidonic acid (AA) at an IC50 of less than 2.1 x 10(-8) M. Vapiprost did not inhibit the primary aggregation or ATP release of human platelets stimulated with adenosine 5'-diphosphate (ADP), epinephrine (Epi) or platelet activating factor (PAF), but inhibited the secondary aggregation stimulated with those agonists at an IC50 of less than 1.3 x 10(-7) M. The sensitivity of platelets in various species of animals to vapiprost was in the following order: human > or = guinea pigs > rats > rabbits. In ex vivo experiments with guinea pigs which received a single oral dose of vapiprost, the agent demonstrated strong inhibition of ATP release from platelets stimulated with U-46619, collagen or AA at an ID50 of less than 25.8 micrograms/kg. These inhibitory effects were observed within 30 min and sustained for 24 h at a single dosage of 5 mg/kg of vapiprost. In AA-induced pulmonary infarction models of mice, the sudden death rates decreased significantly with the oral administration of 10 mg/kg or more of vapiprost. These results indicate that vapiprost effectively inhibits the secondary aggregation and ATP release of human platelets stimulated with various agonists, and that guinea pig and human platelets are similar in response to vapiprost. Furthermore, it was demonstrated in ex vivo experiments with guinea pigs that the inhibitory action of vapiprost appears rapidly and lasts for long periods.

Human trophoblast-derived hydrogen sulfide stimulates placental artery endothelial cell angiogenesis.

PubMed

Chen, Dong-Bao; Feng, Lin; Hodges, Jennifer K; Lechuga, Thomas J; Zhang, Honghai

2017-09-01

Endogenous hydrogen sulfide (H2S), mainly synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), has been implicated in regulating placental angiogenesis; however, the underlying mechanisms are unknown. This study was to test a hypothesis that trophoblasts synthesize H2S to promote placental angiogenesis. Human choriocarcinoma-derived BeWo cells expressed both CBS and CTH proteins, while the first trimester villous trophoblast-originated HTR-8/SVneo cells expressed CTH protein only. The H2S producing ability of BeWo cells was significantly inhibited by either inhibitors of CBS (carboxymethyl hydroxylamine hemihydrochloride, CHH) or CTH (β-cyano-L-alanine, BCA) and that in HTR-8/SVneo cells was inhibited by CHH only. H2S donors stimulated cell proliferation, migration, and tube formation in ovine placental artery endothelial cells (oFPAECs) as effectively as vascular endothelial growth factor. Co-culture with BeWo and HTR-8/SVneo cells stimulated oFPAEC migration, which was inhibited by CHH or BCA in BeWo but CHH only in HTR-8/SVneo cells. Primary human villous trophoblasts (HVT) were more potent than trophoblast cell lines in stimulating oFPAEC migration that was inhibited by CHH and CHH/BCA combination in accordance with its H2S synthesizing activity linked to CBS and CTH expression patterns. H2S donors activated endothelial nitric oxide synthase (NOS3), v-AKT murine thymoma viral oncogene homolog 1 (AKT1), and extracellular signal-activated kinase 1/2 (mitogen-activated protein kinase 3/1, MAPK3/1) in oFPAECs. H2S donor-induced NOS3 activation was blocked by AKT1 but not MAPK3/1 inhibition. In keeping with our previous studies showing a crucial role of AKT1, MAPK3/1, and NOS3/NO in placental angiogenesis, these data show that trophoblast-derived endogenous H2S stimulates placental angiogenesis, involving activation of AKT1, NOS3/NO, and MAPK3/1. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells.

PubMed

Karlsson, Mattias; Scherbak, Nikolai; Khalaf, Hazem; Olsson, Per-Erik; Jass, Jana

2012-11-01

Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Granulocyte Colony-Stimulating Factor and Azole Antifungal Therapy in Murine Aspergillosis: Role of Immune Suppression

PubMed Central

Graybill, John R.; Bocanegra, Rosie; Najvar, Laura K.; Loebenberg, David; Luther, Mike F.

1998-01-01

Outbred ICR mice were immune suppressed either with hydrocortisone or with 5-fluorouracil and were infected intranasally with Aspergillus fumigatus. Beginning 3 days before infection some groups of mice were given recombinant human granulocyte colony-stimulating factor (G-CSF), SCH56592 (an antifungal triazole), or both. Corticosteroid-pretreated mice responded to SCH56592 and had reduced counts in lung tissue and prolonged survival. In these mice, G-CSF strongly antagonized the antifungal activity of SCH56592. Animals treated with both agents developed large lung abscesses with polymorphonuclear leukocytes and large amounts of Aspergillus. In contrast, mice made neutropenic with 5-fluorouracil and then infected with A. fumigatus conidia benefited from either G-CSF or triazoles, and the effect of the combination was additive rather than antagonistic. Host predisposing factors contribute in different ways to the outcome of growth factor therapy in aspergillosis. PMID:9756743

Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

PubMed Central

Hormdee, D; Nagasawa, T; Kiji, M; Yashiro, R; Kobayashi, H; Koshy, G; Noguchi, K; Nitta, H; Ishikawa, I

2005-01-01

Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1α with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E2 (PGE2) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1α-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1α-induced OPG mRNA expression and OPG production. Endogenous PGE2 further enhanced IL-1α-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1α. IL-1α induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1α-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1α-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process. PMID:16297161

Oxalobacter formigenes–Derived Bioactive Factors Stimulate Oxalate Transport by Intestinal Epithelial Cells

PubMed Central

Arvans, Donna; Jung, Yong-Chul; Antonopoulos, Dionysios; Koval, Jason; Granja, Ignacio; Bashir, Mohamed; Karrar, Eltayeb; Roy-Chowdhury, Jayanta; Musch, Mark; Asplin, John; Chang, Eugene

2017-01-01

Hyperoxaluria is a major risk factor for kidney stones and has no specific therapy, although Oxalobacter formigenes colonization is associated with reduced stone risk. O. formigenes interacts with colonic epithelium and induces colonic oxalate secretion, thereby reducing urinary oxalate excretion, via an unknown secretagogue. The difficulties in sustaining O. formigenes colonization underscore the need to identify the derived factors inducing colonic oxalate secretion. We therefore evaluated the effects of O. formigenes culture conditioned medium (CM) on apical 14C-oxalate uptake by human intestinal Caco-2-BBE cells. Compared with control medium, O. formigenes CM significantly stimulated oxalate uptake (>2.4-fold), whereas CM from Lactobacillus acidophilus did not. Treating the O. formigenes CM with heat or pepsin completely abolished this bioactivity, and selective ultrafiltration of the CM revealed that the O. formigenes–derived factors have molecular masses of 10–30 kDa. Treatment with the protein kinase A inhibitor H89 or the anion exchange inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid completely blocked the CM-induced oxalate transport. Knockdown of the oxalate transporter SLC26A6 also significantly restricted the induction of oxalate transport by CM. In a mouse model of primary hyperoxaluria type 1, rectal administration of O. formigenes CM significantly reduced (>32.5%) urinary oxalate excretion and stimulated (>42%) distal colonic oxalate secretion. We conclude that O. formigenes–derived bioactive factors stimulate oxalate transport in intestinal cells through mechanisms including PKA activation. The reduction in urinary oxalate excretion in hyperoxaluric mice treated with O. formigenes CM reflects the in vivo retention of biologic activity and the therapeutic potential of these factors. PMID:27738124

Angiogenic CXC chemokine expression during differentiation of human mesenchymal stem cells towards the osteoblastic lineage.

PubMed

Bischoff, D S; Zhu, J H; Makhijani, N S; Kumar, A; Yamaguchi, D T

2008-02-15

The potential role of ELR(+) CXC chemokines in early events in bone repair was studied using human mesenchymal stem cells (hMSCs). Inflammation, which occurs in the initial phase of tissue healing in general, is critical to bone repair. Release of cytokines from infiltrating immune cells and injured bone can lead to recruitment of MSCs to the region of repair. CXC chemokines bearing the Glu-Leu-Arg (ELR) motif are also released by inflammatory cells and serve as angiogenic factors stimulating chemotaxis and proliferation of endothelial cells. hMSCs, induced to differentiate with osteogenic medium (OGM) containing ascorbate, beta-glycerophosphate (beta-GP), and dexamethasone (DEX), showed an increase in mRNA and protein secretion of the ELR(+) CXC chemokines CXCL8 and CXCL1. CXCL8 mRNA half-life studies reveal an increase in mRNA stability upon OGM stimulation. Increased expression and secretion is a result of DEX in OGM and is dose-dependent. Inhibition of the glucocorticoid receptor with mifepristone only partially inhibits DEX-stimulated CXCL8 expression indicating both glucocorticoid receptor dependent and independent pathways. Treatment with signal transduction inhibitors demonstrate that this expression is due to activation of the ERK and p38 mitogen-activated protein kinase (MAPK) pathways and is mediated through the G(alphai)-coupled receptors. Angiogenesis assays demonstrate that OGM-stimulated conditioned media containing secreted CXCL8 and CXCL1 can induce angiogenesis of human microvascular endothelial cells in an in vitro Matrigel assay. Copyright 2007 Wiley-Liss, Inc.

Granulocyte-macrophage colony-stimulating factor amplification of interleukin-1beta and tumor necrosis factor alpha production in THP-1 human monocytic cells stimulated with lipopolysaccharide of oral microorganisms.

PubMed

Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

1998-05-01

Cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF), are used to assist in bone marrow recovery during cancer chemotherapy. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) play important roles in inflammatory processes, including exacerbation of periodontal diseases, one of the most common complications in patients who undergo this therapy. A human monocyte cell line (THP-1) was utilized to investigate IL-1beta and TNF-alpha production following GM-CSF supplementation with lipopolysaccharide (LPS) from two oral microorganisms, Porphyromonas gingivalis and Fusobacterium nucleatum. LPS of P. gingivalis or F. nucleatum was prepared by a phenol-water extraction method and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and determination of total protein and endotoxin contents. Resting THP-1 cells were treated with LPS of P. gingivalis or F. nucleatum and/or GM-CSF (50 IU/ml) by using different concentrations for various time periods. Production of IL-1beta and TNF-alpha in THP-1 cells was measured by solid-phase enzyme-linked immunosorbent assay. Reverse transcription (RT)-PCR was used to evaluate the gene expression of resting and treated THP-1 cells. IL-1beta was not detected in untreated THP-1 cells. IL-1beta production was, however, stimulated sharply at 4 h. GM-CSF amplified IL-1beta production in THP-1 cells treated with LPS from both oral anaerobes. No IL-1beta-specific mRNA transcript was detected in untreated THP-1 cells. However, IL-1beta mRNA was detected by RT-PCR 2 h after stimulation of THP-1 cells with LPS from both organisms. GM-CSF did not shorten the IL-1beta transcriptional activation time. GM-CSF plus F. nucleatum or P. gingivalis LPS activated THP-1 cells to produce a 1.6-fold increase in TNF-alpha production at 4 h over LPS stimulation alone. These investigations with the in vitro THP-1 model indicate that there may be an increase in the cellular immune response to oral endotoxin following GM-CSF therapy, as evidenced by production of the tissue-reactive cytokines IL-1beta and TNF-alpha.

RON kinase inhibition reduces renal endothelial injury in sickle cell disease mice

PubMed Central

Khaibullina, Alfia; Adjei, Elena A.; Afangbedji, Nowah; Ivanov, Andrey; Kumari, Namita; Almeida, Luis E.F.; Quezado, Zenaide M.N.; Nekhai, Sergei; Jerebtsova, Marina

2018-01-01

Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression of Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of the development of renal disease in sickle cell disease. PMID:29519868

Reversal of cigarette smoke extract-induced sinonasal epithelial cell barrier dysfunction through Nrf2 Activation.

PubMed

Tharakan, Anuj; Halderman, Ashleigh A; Lane, Andrew P; Biswal, Shyam; Ramanathan, Murugappan

2016-11-01

Environmental factors such as inhaled pollutants like cigarette smoke may play a significant role in diseases of the upper airway including chronic rhinosinusitis (CRS). Recent studies have shown that cigarette smoke causes impaired airway epithelial cell barrier function likely through environmental oxidative stress related pathways. The purpose of this study is to explore whether enhancing nuclear factor erythroid 2 [NF-E2]-related factor 2 [Nrf2], the body's master antioxidant system, can ameliorate cigarette smoke-induced sinonasal epithelial cell (SNEC) barrier dysfunction. Human SNECs (HSNECs) were grown from control patients at the air-liquid interface (ALI). HSNECs were stimulated with cigarette smoke extract (CSE) with and without pharmacologic activation of Nrf2. HSNECs were then stained for the epithelial cell junctional proteins zonula occludens 1 (ZO-1) and junctional adhesion molecule A (JAM-A) using confocal microscopy. In addition, transepithelial electrical resistance (TER) was measured in cultures before and after stimulation with CSE. CSE stimulation caused a global disruption of the epithelial junctional proteins ZO-1 and JAM-A along with an associated decrease in TER levels. Enhancing Nrf2 levels prior to stimulation with CSE was associated with increased localization of ZO-1 and JAM-A levels at the cell surface and statistically significant increases in TER levels. This is the first study to demonstrate that cigarette smoke induced SNEC barrier dysfunction is reversible by Nrf2 activation. The Nrf2 antioxidant pathway may represent a potential therapeutic target for cigarette smoke-associated sinonasal inflammation. © 2016 ARS-AAOA, LLC.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Human Performance in Continuous/Sustained Operations and the Demands of Extended Work/Rest Schedules: An Annotated Bibliography. Volume 2

DTIC Science & Technology

1989-06-01

subjective self -report questionnaires and more objective activity measures to estimate the amount of sleep obtained. The aim of the study was to determine...helicopter crews were observed and analyzed. It is concluded that the operational effiency of flight crews can be obtained by adopting four measures...task on simulated truck- driving task performance. Human factors. 27(2): 201-207. A study examined the effects of extra task stimulation and extra

An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

1998-04-01

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Differential Transcription Factor Use by the KIR2DL4 Promoter Under Constitutive and IL-2/15-Treated Conditions

PubMed Central

Presnell, Steven R.; Zhang, Lei; Chlebowy, Corrin N.; Al-Attar, Ahmad; Lutz, Charles T.

2012-01-01

KIR2DL4 is unique among human KIR genes in expression, cellular localization, structure, and function, yet the transcription factors required for its expression have not been identified. Using mutagenesis, electrophoretic mobility shift assay, and co-transfection assays, we identified two redundant Runx binding sites in the 2DL4 promoter as essential for constitutive 2DL4 transcription, with contributions by a CRE site and initiator elements. IL-2-and IL-15-stimulated human NK cell lines increased 2DL4 promoter activity, which required functional Runx, CRE, and Ets sites. Chromatin immunoprecipitation experiments show that Runx3 and Ets1 bind the 2DL4 promoter in situ. 2DL4 promoter activity had similar transcription factor requirements in T cells. Runx, CRE, and Ets binding motifs are present in 2DL4 promoters from across primate species, but other postulated transcription factor binding sites are not preserved. Differences between 2DL4 and clonally-restricted KIR promoters suggest a model that explains the unique 2DL4 expression pattern in human NK cells. PMID:22467658

Characterization of a human antigen specific helper factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richardson, B.

1986-03-01

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with /sup 35/S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEMmore » line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants.« less

DNA activates human immune cells through a CpG sequence-dependent manner

PubMed Central

Bauer, M; Heeg, K; Wagner, H; Lipford, G B

1999-01-01

While bacterial DNA and cytosine–guanosine-dinucleotide-containing oligonucleotides (CpG ODN) are well described activators of murine immune cells, their effect on human cells is inconclusive. We investigated their properties on human peripheral blood mononuclear cells (PBMC) and subsets thereof, such as purified monocytes, T and B cells. Here we demonstrate that bacterial DNA and CpG ODN induce proliferation of B cells, while other subpopulations, such as monocytes and T cells, did not proliferate. PBMC mixed cell cultures, as well as purified monocytes, produced interleukin-6 (IL-6), IL-12 and tumour necrosis factor-α upon stimulation with bacterial DNA; however, only IL-6 and IL-12 secretion became induced upon CpG ODN stimulation. We conclude that monocytes, but not B or T cells, represent the prime source of cytokines. Monocytes up-regulated expression of antigen-presenting, major histocompatibility complex class I and class II molecules in response to CpG DNA. In addition, both monocytes and B cells up-regulate costimulatory CD86 and CD40 molecules. The activation by CpG ODN depended on sequence motifs containing the core dinucleotide CG since destruction of the motif strongly reduced immunostimulatory potential. PMID:10457226

Identification of a major 50-kDa molecular weight human B-cell growth factor with Tac antigen-inducing activity on B cells.

PubMed

Kawano, M; Matsushima, K; Oppenheim, J J

1987-08-01

A bioassay was developed using human small B cells adherent to anti-human IgM (anti-mu)-coated wells. These B cells were stimulated to proliferate by culture supernatants of concanavalin A (Con A)-activated human peripheral blood lymphocytes (Con A Sup) even in the presence of high concentrations of anti-mu coated on assay wells. Human B-cell growth factor (BCGF) activities were partially purified from Con A Sup. Preparative chromatography (Sephacryl S-200 and isoelectrofocusing) yielded a major peak of BCGF activity for B cells adherent to anti-mu-coated wells with a molecular weight of 50,000 (50 kDa) and a pI 7.6. The 50-kDa BCGF was further purified by sequential chromatography using DEAE-Sephacel, CM-Sepharose, Sephacryl S-200, CM-high performance liquid chromatography (HPLC), and hydroxyapatite (HA)-HPLC. The HA-HPLC-purified 50-kDa BCGF was free of interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon activities, but could support growth of BCL1 cells, similar to BCGF-II. Neither IL-1 nor interferon-gamma had any growth-stimulating effect in our B-cell proliferation assay with or without BCGF in Iscove's synthetic assay medium. BCGF-induced proliferation of B cells adherent to anti-mu-coated wells could be markedly augmented by the simultaneous or sequential addition of recombinant human IL-2 (rIL-2). When cultured for 3 days with 50-kDa BCGF, about 40% of B cells adherent to anti-mu-coated wells expressed Tac antigen, and monoclonal anti-Tac antibody inhibited rIL-2 enhancement of proliferation of 50-kDa BCGF-preactivated B cells. In addition, 50-kDa BCGF could induce Tac antigen on an Epstein-Barr virus-transformed B-cell line (ORSON) in the presence of a suboptimal dose of phorbol myristate acetate (PMA) and also on a natural killer-like cell line (YT cells). We have therefore identified a major 50-kDa BCGF activity with Tac antigen-inducing activity that also has a synergistic effect with IL-2 on normal B-cell proliferation.

Receptor for macrophage colony-stimulating factor transduces a signal decreasing erythroid potential in the multipotent hematopoietic EML cell line.

PubMed

Pawlak, G; Grasset, M F; Arnaud, S; Blanchet, J P; Mouchiroud, G

2000-10-01

To test the hypothesis that hematopoietic growth factors may influence lineage choice in pluripotent progenitor cells, we investigated the effects of macrophage colony-stimulating factor (M-CSF) on erythroid and myeloid potentials of multipotent EML cells ectopically expressing M-CSF receptor (M-CSFR). EML cells are stem cell factor (SCF)-dependent murine cells that give rise spontaneously to pre-B cells, burst-forming unit erythroid (BFU-E), and colony-forming unit granulocyte macrophage (CFU-GM). We determined BFU-E and CFU-GM frequencies among EML cells transduced with murine M-CSFR, human M-CSFR, or chimeric receptors, and cultivated in the presence of SCF, M-CSF, or both growth factors. Effects of specific inhibitors of signaling molecules were investigated. EML cells transduced with murine M-CSFR proliferated in response to M-CSF but also exhibited a sharp and rapid decrease in BFU-E frequency associated with an increase in CFU-GM frequency. In contrast, EML cells expressing human M-CSFR proliferated in response to M-CSF without any changes in erythroid or myeloid potential. Using chimeric receptors between human and murine M-CSFR, we showed that the effects of M-CSF on EML cell differentiation potential are mediated by a large region in the intracellular domain of murine M-CSFR. Furthermore, phospholipase C (PLC) inhibitor U73122 interfered with the negative effects of ligand-activated murine M-CSFR on EML cell erythroid potential. We propose that signaling pathways activated by tyrosine kinase receptors may regulate erythroid potential and commitment decisions in multipotent progenitor cells and that PLC may play a key role in this process.

Control of Human Endometrial Stromal Cell Motility by PDGF-BB, HB-EGF and Trophoblast-Secreted Factors

PubMed Central

Schwenke, Maren; Knöfler, Martin; Velicky, Philipp; Weimar, Charlotte H. E.; Kruse, Michelle; Samalecos, Annemarie; Wolf, Anja; Macklon, Nick S.; Bamberger, Ana-Maria; Gellersen, Birgit

2013-01-01

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site. PMID:23349855

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

PubMed

Sugatani, T; Alvarez, U M; Hruska, K A

2003-09-01

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis. Copyright 2003 Wiley-Liss, Inc.

Activin-A as an intraovarian modulator: actions, localization, and regulation of the intact dimer in human ovarian cells.

PubMed Central

Rabinovici, J; Spencer, S J; Doldi, N; Goldsmith, P C; Schwall, R; Jaffe, R B

1992-01-01

The actions, localization, and regulation of activin in the human ovary are unknown. Therefore, the aims of this study were (a) to define the effects of recombinant activin-A and its structural homologue, inhibin-A, on mitogenesis and steroidogenesis (progesterone secretion and aromatase activity) in human preovulatory follicular cells; (b) to localize the activin-A dimer in the human ovary by immunohistochemistry; and (c) to examine regulation of intracellular activin-A production in cultured human follicular cells. In addition to stimulating mitogenic activity, activin-A causes a dose- and time-dependent inhibition of basal and gonadotropin-stimulated progesterone secretion and aromatase activity in human luteinizing follicular cells on day 2 and day 4 of culture. Inhibin-A exerts no effects on mitogenesis, basal or gonadotropin-stimulated progesterone secretion and aromatase activity, and does not alter effects observed with activin-A alone. Immunostaining for dimeric activin-A occurs in granulosa and cumulus cells of human ovarian follicles and in granulosa-lutein cells of the human corpus luteum. cAMP, and to a lesser degree human chorionic gonadotropin and follicle-stimulating hormone, but not inhibin-A, activin-A, or phorbol 12-myristate 13-acetate, increased the immunostaining for activin-A in cultured granulosa cells. These results indicate that activin-A may function as an autocrine or paracrine regulator of follicular function in the human ovary. Images PMID:1569191

Porcine endothelium induces DNA-histone complex formation in human whole blood: a harmful effect of histone on coagulation and endothelial activation.

PubMed

Yoo, Hyun Ju; Kim, Ji-Eun; Gu, Ja Yoon; Lee, Sae Bom; Lee, Hyun Joo; Hwang, Ho Young; Hwang, Yoohwa; Kim, Young Tae; Kim, Hyun Kyung

2016-11-01

Neutrophils play a role in xenograft rejection. When neutrophils are stimulated, they eject the DNA-histone complex into the extracellular space, called neutrophil extracellular traps (NET). We investigated whether NET formation actively occurs in the xenograft and contributes to coagulation and endothelial activation. Human whole blood was incubated with porcine aortic endothelial cells (pEC) from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. In the supernatant plasma from human blood, the level of the DNA-histone complex was measured by ELISA, and thrombin generation was measured using a calibrated automated thrombogram. Histone-induced tissue factor and adhesion molecule expression were measured by flow cytometry. pEC from both wild-type and GTKO pigs significantly induced DNA-histone complex formation in human whole blood. The DNA-histone complex produced shortened the thrombin generation time and clotting time. Histone alone dose-dependently induced tissue factor and adhesion molecule expression in pEC. Aurintricarboxylic acid pretreatment partially inhibited pEC-induced DNA-histone complex formation. DNA-histone complex actively generated upon xenotransplantation is a novel target to inhibit coagulation and endothelial activation. To prevent tissue factor and adhesion molecule expression, a strategy to block soluble histone may be required in xenotransplantation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Attentional load and sensory competition in human vision: modulation of fMRI responses by load at fixation during task-irrelevant stimulation in the peripheral visual field.

PubMed

Schwartz, Sophie; Vuilleumier, Patrik; Hutton, Chloe; Maravita, Angelo; Dolan, Raymond J; Driver, Jon

2005-06-01

Perceptual suppression of distractors may depend on both endogenous and exogenous factors, such as attentional load of the current task and sensory competition among simultaneous stimuli, respectively. We used functional magnetic resonance imaging (fMRI) to compare these two types of attentional effects and examine how they may interact in the human brain. We varied the attentional load of a visual monitoring task performed on a rapid stream at central fixation without altering the central stimuli themselves, while measuring the impact on fMRI responses to task-irrelevant peripheral checkerboards presented either unilaterally or bilaterally. Activations in visual cortex for irrelevant peripheral stimulation decreased with increasing attentional load at fixation. This relative decrease was present even in V1, but became larger for successive visual areas through to V4. Decreases in activation for contralateral peripheral checkerboards due to higher central load were more pronounced within retinotopic cortex corresponding to 'inner' peripheral locations relatively near the central targets than for more eccentric 'outer' locations, demonstrating a predominant suppression of nearby surround rather than strict 'tunnel vision' during higher task load at central fixation. Contralateral activations for peripheral stimulation in one hemifield were reduced by competition with concurrent stimulation in the other hemifield only in inferior parietal cortex, not in retinotopic areas of occipital visual cortex. In addition, central attentional load interacted with competition due to bilateral versus unilateral peripheral stimuli specifically in posterior parietal and fusiform regions. These results reveal that task-dependent attentional load, and interhemifield stimulus-competition, can produce distinct influences on the neural responses to peripheral visual stimuli within the human visual system. These distinct mechanisms in selective visual processing may be integrated within posterior parietal areas, rather than earlier occipital cortex.

Cosmos-1989 immunology studies

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald

1991-01-01

Evidence from both human and rodent studies has indicated that alterations in immunological parameters occur after space flight. The number of flight experiments has been small, and the full breadth of immunological alterations occurring after space flight remains to be established. Among the major effects on immune responses after space flight that have been reported are: alterations in lymphocyte blastogenesis and natural killer cell activity, alterations in production of cytokines, changes in leukocyte sub-population distribution, and decreases in the ability in the ability of bone marrow cells to respond to colony stimulating factors. Changes have been reported in immunological parameters of both humans and rodents. The significance of these alterations in relation to resistance to infection remains to be established. The current study involved a determination of the effects of flight on Cosmos mission 2044 on leukocyte subset distribution and the sensitivity of bone marrow cells to colony stimulating factor-GM. A parallel study with antiorthostatic suspension was also carried out. The study involved repetition and expansion of studies carried out on Cosmos 1887.

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

PubMed

Murata, H; Hattori, T; Maeda, H; Takashiba, S; Takigawa, M; Kido, J; Nagata, T

2015-08-01

Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. We identified TDP-43 as one of the novel TNF-α factors and found that it bound to the LPS-responsive element in the TNF-α promoter to increase TNF-α expression. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Estrogen sulfotransferases in breast and endometrial cancers.

PubMed

Pasqualini, Jorge Raul

2009-02-01

Estrogen sulfotransferase is significantly more active in the normal breast cell (e.g., Human 7) than in the cancer cell (e.g., MCF-7). The data suggest that in breast cancer sulfoconjugated activity is carried out by another enzyme, the SULT1A, which acts at high concentration of the substrates. In breast cancer cells sulfotransferase (SULT) activity can be stimulated by various progestins: medrogestone, promegestone, and nomegestrol acetate, as well as by tibolone and its metabolites. SULT activities can also be controlled by other substances including phytoestrogens, celecoxib, flavonoids (e.g., quercetin, resveratrol), and isoflavones. SULT expression was localized in breast cancer cells, which can be stimulated by promegestone and correlated with the increase of the enzyme activity. The estrogen sulfotransferase (SULT1E1), which acts at nanomolar concentration of estradiol, can inactivate most of this hormone present in the normal breast; however, in the breast cancer cells, the sulfotransferase denoted as SULT1A1 is mainly present, and this acts at micromolar concentrations of E(2). A correlation was postulated among breast cancer cell proliferation, the effect of various progestins, and sulfotransferase stimulation. In conclusion, it is suggested that factors involved in the stimulation of the estrogen sulfotransferases could provide new possibilities for the treatment of patients with hormone-dependent breast and endometrial cancers.

Effects of microgravity on the immune system

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Taylor, Gerald R.

1991-01-01

Changes in resistance to bacterial and viral infections in Apollo crew members has stimulated interest in the study of immunity and space flight. Results of studies from several laboratories in both humans and rodents have indicated alterations after space flight that include the following immunological parameters: thymus size, lymphocyte blastogenesis, interferon and interleukin production, natural killer cell activity, cytotoxic T-cell activity, leukocyte subset population distribution, response of bone marrow cells to colony stimulating factors, and delayed hypersensitivity skin test reactivity. The interactions of the immune system with other physiological systems, including muscle, bone, and the nervous system, may play a major role in the development of these immunological parameters during and after flight. There may also be direct effects of space flight on immune responses.

Daidzein suppresses tumor necrosis factor-α induced migration and invasion by inhibiting hedgehog/Gli1 signaling in human breast cancer cells.

PubMed

Bao, Cheng; Namgung, Hyeju; Lee, Jaehoo; Park, Hyun-Chang; Ko, Jiwon; Moon, Heejung; Ko, Hyuk Wan; Lee, Hong Jin

2014-04-30

In breast cancer, the cytokine tumor necrosis factor-α (TNF-α) induces cell invasion, although the molecular basis of it has not been clearly elucidated. In this study, we investigated the role of daidzein in regulating TNF-α induced cell invasion and the underlying molecular mechanisms. Daidzein inhibited TNF-α induced cellular migration and invasion in estrogen receptor (ER) negative MCF10DCIS.com human breast cancer cells. TNF-α activated Hedgehog (Hh) signaling by enhancing Gli1 nuclear translocation and transcriptional activity, which resulted in increased invasiveness; these effects were blocked by daidzein and the Hh signaling inhibitors, cyclopamine and vismodegib. Moreover, these compounds suppressed TNF-α induced matrix metalloproteinase (MMP)-9 mRNA expression and activity. Taken together, mammary tumor cell invasiveness was stimulated by TNF-α induced activation of Hh signaling; these effects were abrogated by daidzein, which suppressed Gli1 activation, thereby inhibiting migration and invasion.

Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells.

PubMed

Chang, Hebron C; Yang, Hsin-Ling; Pan, Jih-Hao; Korivi, Mallikarjuna; Pan, Jian-You; Hsieh, Meng-Chang; Chao, Pei-Min; Huang, Pei-Jane; Tsai, Ching-Tsan; Hseu, You-Cheng

2016-01-01

Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50-200 μg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways.

Hericium erinaceus Inhibits TNF-α-Induced Angiogenesis and ROS Generation through Suppression of MMP-9/NF-κB Signaling and Activation of Nrf2-Mediated Antioxidant Genes in Human EA.hy926 Endothelial Cells

PubMed Central

Chang, Hebron C.; Yang, Hsin-Ling; Pan, Jih-Hao; Korivi, Mallikarjuna; Pan, Jian-You; Hsieh, Meng-Chang; Chao, Pei-Min; Huang, Pei-Jane; Tsai, Ching-Tsan; Hseu, You-Cheng

2016-01-01

Hericium erinaceus (HE) is an edible mushroom that has been shown to exhibit anticancer and anti-inflammatory activities. We investigated the antiangiogenic and antioxidant potentials of ethanol extracts of HE in human endothelial (EA.hy926) cells upon tumor necrosis factor-α- (TNF-α-) stimulation (10 ng/mL). The underlying molecular mechanisms behind the pharmacological efficacies were elucidated. We found that noncytotoxic concentrations of HE (50–200 μg/mL) significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation of endothelial cells. HE treatment suppressed TNF-α-induced activity and/or overexpression of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). Furthermore, HE downregulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) followed by suppression of I-κB (inhibitor-κB) degradation. Data from fluorescence microscopy illustrated that increased intracellular ROS production upon TNF-α-stimulation was remarkably inhibited by HE pretreatment in a dose-dependent manner. Notably, HE triggered antioxidant gene expressions of heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase (γ-GCLC), and glutathione levels, which may contribute to inhibition of ROS. Increased antioxidant status was associated with upregulated nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2) in HE treated cells. Our findings conclude that antiangiogenic and anti-inflammatory activities of H. erinaceus may contribute to its anticancer property through modulation of MMP-9/NF-κB and Nrf2-antioxidant signaling pathways. PMID:26823953

Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis.

PubMed

Komaki, Motohiro; Numata, Yuri; Morioka, Chikako; Honda, Izumi; Tooi, Masayuki; Yokoyama, Naoki; Ayame, Hirohito; Iwasaki, Kengo; Taki, Atsuko; Oshima, Noriko; Morita, Ikuo

2017-10-03

The therapeutic potential of mesenchymal stem cells (MSCs) may be attributed partly to humoral factors such as growth factors, cytokines, and chemokines. Human term placental tissue-derived MSCs (PlaMSCs), or conditioned medium left over from cultures of these cells, have been reported to enhance angiogenesis. Recently, the exosome, which can transport a diverse suite of macromolecules, has gained attention as a novel intercellular communication tool. However, the potential role of the exosome in PlaMSC therapeutic action is not well understood. The purpose of this study was to evaluate PlaMSC-derived exosome angiogenesis promotion in vitro and in vivo. MSCs were isolated from human term placental tissue by enzymatic digestion. Conditioned medium was collected after 48-h incubation in serum-free medium (PlaMSC-CM). Angiogenic factors present in PlaMSC-CM were screened by a growth factor array. Exosomes were prepared by ultracentrifugation of PlaMSC-CM, and confirmed by transmission electron microscopy, dynamic light scattering, and western blot analyses. The proangiogenic activity of PlaMSC-derived exosomes (PlaMSC-exo) was assessed using an endothelial tube formation assay, a cell migration assay, and reverse transcription-PCR analysis. The in-vivo angiogenic activity of PlaMSC-exo was evaluated using a murine auricle ischemic injury model. PlaMSC-CM contained both angiogenic and angiostatic factors, which enhanced endothelial tube formation. PlaMSC-exo were incorporated into endothelial cells; these exosomes stimulated both endothelial tube formation and migration, and enhanced angiogenesis-related gene expression. Laser Doppler blood flow analysis showed that PlaMSC-exo infusion also enhanced angiogenesis in an in-vivo murine auricle ischemic injury model. PlaMSC-exo enhanced angiogenesis in vitro and in vivo, suggesting that exosomes play a role in the proangiogenic activity of PlaMSCs. PlaMSC-exo may be a novel therapeutic approach for treating ischemic diseases.

Reducing the Disruptive Effects of Interruptions With Noninvasive Brain Stimulation.

PubMed

Blumberg, Eric J; Foroughi, Cyrus K; Scheldrup, Melissa R; Peterson, Matthew S; Boehm-Davis, Debbie A; Parasuraman, Raja

2015-09-01

The authors determine whether transcranial direct current stimulation (tDCS) can reduce resumption time when an ongoing task is interrupted. Interruptions are common and disruptive. Working memory capacity has been shown to predict resumption lag (i.e., time to successfully resume a task after interruption). Given that tDCS applied to brain areas associated with working memory can enhance performance, tDCS has the potential to improve resumption lag when a task is interrupted. Participants were randomly assigned to one of four groups that received anodal (active) stimulation of 2 mA tDCS to one of two target brain regions, left and right dorsolateral prefrontal cortex (DLPFC), or to one of two control areas, active stimulation of the left primary motor cortex or sham stimulation of the right DLPFC, while completing a financial management task that was intermittently interrupted with math problem solving. Anodal stimulation to the right and left DLPFC significantly reduced resumption lags compared to the control conditions (sham and left motor cortex stimulation). Additionally, there was no speed-accuracy tradeoff (i.e., the improvement in resumption time was not accompanied by an increased error rate). Noninvasive brain stimulation can significantly decrease resumption lag (improve performance) after a task is interrupted. Noninvasive brain stimulation offers an easy-to-apply tool that can significantly improve interrupted task performance. © 2014, Human Factors and Ergonomics Society.

Nutritionally relevant concentrations of resveratrol and hydroxytyrosol mitigate oxidative burst of human granulocytes and monocytes and the production of pro-inflammatory mediators in LPS-stimulated RAW 264.7 macrophages.

PubMed

Bigagli, Elisabetta; Cinci, Lorenzo; Paccosi, Sara; Parenti, Astrid; D'Ambrosio, Mario; Luceri, Cristina

2017-02-01

The health benefits of bio-active phenolic compounds have been largely investigated in vitro at concentrations which exceed those reachable in vivo. We investigated and compared the anti-inflammatory effects of resveratrol, hydroxytyrosol and oleuropein at physiologically relevant concentrations by using in vitro models of inflammation. Human granulocytes and monocytes were stimulated with phorbol myristate acetate (PMA) and the ability of resveratrol, hydroxytyrosol and oleuropein to inhibit the oxidative burst and CD11b expression was measured. Nitric oxide (NO), prostaglandin E2 (PGE2) levels, COX-2, iNOS, TNFα, IL-1β and miR-146a expression and activation of the transcription factor Nrf2 were evaluated in macrophages RAW 264.7 stimulated with LPS (1μg/ml) for 18h, exposed to resveratrol, hydroxytyrosol and oleuropein (5 and 10μM). Synergistic effects were explored as well, together with the levels of PGE2, COX-2 and IL-1β expression in macrophages after 6h of LPS stimulation. PGE2 and COX-2 expression were also assessed on human monocytes. All the tested compounds inhibited granulocytes oxidative burst in a concentration dependent manner and CD11b expression was also significantly counteracted by resveratrol and hydroxytyrosol. The measurement of oxidative burst in human monocytes produced similar effects being resveratrol more active. Hydroxytyrosol and resveratrol inhibited the production of NO and PGE2 but did not reduce iNOS, TNFα or IL-1β gene expression in LPS-stimulated RAW 264.7 for 18h. Resveratrol slightly decreased COX-2 expression after 18h but not after 6h, but reduced PGE2 levels after 6h. Resveratrol and hydroxytyrosol 10μM induced NRf2 nuclear translocation and reduced miR-146a expression in LPS treated RAW 264.7. Overall, we reported an anti-inflammatory effect of resveratrol and hydroxytyrosol at low, nutritionally relevant concentrations, involving the inhibition of granulocytes and monocytes activation, the modulation of miR-146a expression and the activation of Nrf2. A regular dietary intake of resveratrol and hydroxytyrosol may be a useful complementary strategy to control inflammatory diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guyda, H.J.

1991-03-01

The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGF-II were determined on human placental cells in monolayer culture obtained from early gestation (less than 20 weeks) and late gestation (38-42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and (3H)thymidine incorporation into cell protein. Since benzo(alpha)pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo(alpha)pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured humanmore » placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10-100 micrograms/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at a high concentration in both early and late gestation cells, suggesting an action via type 1 IGF receptors rather than via insulin receptors. EGF stimulated 3-O-methylglucose uptake only in term placental cells. No significant stimulation of (3H)thymidine incorporation by any of the growth factors tested was seen with either early or late gestation cells. The effect of PAHs on AIB uptake by cultured placental cells was variable. BP alone stimulated AIB uptake by both very early and late gestation cells and enhanced EGF-stimulated AIB uptake. alpha-naphthoflavone alone inhibited AIB uptake at all gestational ages and inhibited EGF-stimulated AIB uptake. beta-Naphthoflavone and 3-methylcholanthrene minimally inhibited AIB uptake by early gestation cells and did not modify EGF-stimulated uptake at any gestational period.« less

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells.

PubMed

Huang, F-M; Chen, Y-J; Chou, M-Y; Chang, Y-C

2005-12-01

To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

Gadolinium compounds signaling through TLR4 and TLR7 in normal human macrophages: establishment of a proinflammatory phenotype and implications for the pathogenesis of nephrogenic systemic fibrosis.

PubMed

Wermuth, Peter J; Jimenez, Sergio A

2012-07-01

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity pathways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-κB, and increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-κB-inducible reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-1R-associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression. Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-6, TGF-β, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis.

DNA from Periodontopathogenic Bacteria Is Immunostimulatory for Mouse and Human Immune Cells

PubMed Central

Nonnenmacher, Claudia; Dalpke, Alexander; Zimmermann, Stefan; Flores-de-Jacoby, Lavin; Mutters, Reinier; Heeg, Klaus

2003-01-01

Although bacterial DNA (bDNA) containing unmethylated CpG motifs stimulates innate immune cells through Toll-like receptor 9 (TLR-9), its precise role in the pathophysiology of diseases is still equivocal. Here we examined the immunostimulatory effects of DNA extracted from periodontopathogenic bacteria. A major role in the etiology of periodontal diseases has been attributed to Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Peptostreptococcus micros. We therefore isolated DNA from these bacteria and stimulated murine macrophages and human gingival fibroblasts (HGF) in vitro. Furthermore, HEK 293 cells transfected with human TLR-9 were also stimulated with these DNA preparations. We observed that DNA from these pathogens stimulates macrophages and gingival fibroblasts to produce tumor necrosis factor alpha and interleukin-6 in a dose-dependent manner. Methylation of the CpG motifs abolished the observed effects. Activation of HEK 293 cells expressing TLR-9 which were responsive to bDNA but not to lipopolysaccharide confirmed that immunostimulation was achieved by bDNA. In addition, the examined bDNA differed in the ability to stimulate murine macrophages, HGF, and TLR-9-transfected cells. DNA from A. actinomycetemcomitans elicited a potent cytokine response, while DNA from P. gingivalis and P. micros showed lower immunostimulatory activity. Taken together, the results strongly suggest that DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros possesses immunostimulatory properties in regard to cytokine secretion by macrophages and fibroblasts. These stimulatory effects are due to unmethylated CpG motifs within bDNA and differ between distinct periodontopathogenic bacteria strains. Hence, immunostimulation by DNA from A. actinomycetemcomitans, P. gingivalis, and P. micros could contribute to the pathogenesis of periodontal diseases. PMID:12540566

Stimulation of osteogenic differentiation in human osteoprogenitor cells by pulsed electromagnetic fields: an in vitro study

PubMed Central

2010-01-01

Background Although pulsed electromagnetic field (PEMF) stimulation may be clinically beneficial during fracture healing and for a wide range of bone disorders, there is still debate on its working mechanism. Mesenchymal stem cells are likely mediators facilitating the observed clinical effects of PEMF. Here, we performed in vitro experiments to investigate the effect of PEMF stimulation on human bone marrow-derived stromal cell (BMSC) metabolism and, specifically, whether PEMF can stimulate their osteogenic differentiation. Methods BMSCs derived from four different donors were cultured in osteogenic medium, with the PEMF treated group being continuously exposed to a 15 Hz, 1 Gauss EM field, consisting of 5-millisecond bursts with 5-microsecond pulses. On culture day 1, 5, 9, and 14, cells were collected for biochemical analysis (DNA amount, alkaline phosphatase activity, calcium deposition), expression of various osteoblast-relevant genes and activation of extracellular signal-regulated kinase (ERK) signaling. Differences between treated and control groups were analyzed using the Wilcoxon signed rank test, and considered significant when p < 0.05. Results Biochemical analysis revealed significant, differentiation stage-dependent, PEMF-induced differences: PEMF increased mineralization at day 9 and 14, without altering alkaline phosphatase activity. Cell proliferation, as measured by DNA amounts, was not affected by PEMF until day 14. Here, DNA content stagnated in PEMF treated group, resulting in less DNA compared to control. Quantitative RT-PCR revealed that during early culture, up to day 9, PEMF treatment increased mRNA levels of bone morphogenetic protein 2, transforming growth factor-beta 1, osteoprotegerin, matrix metalloproteinase-1 and -3, osteocalcin, and bone sialoprotein. In contrast, receptor activator of NF-κB ligand expression was primarily stimulated on day 14. ERK1/2 phosphorylation was not affected by PEMF stimulation. Conclusions PEMF exposure of differentiating human BMSCs enhanced mineralization and seemed to induce differentiation at the expense of proliferation. The osteogenic stimulus of PEMF was confirmed by the up-regulation of several osteogenic marker genes in the PEMF treated group, which preceded the deposition of mineral itself. These findings indicate that PEMF can directly stimulate osteoprogenitor cells towards osteogenic differentiation. This supports the theory that PEMF treatment may recruit these cells to facilitate an osteogenic response in vivo. PMID:20731873

The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

PubMed

Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

2009-04-01

To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P < 0.05). Investigations of the time dependence of RANKL expression in IL-1alpha-treated cells revealed a rapid accumulation of RANKL protein after 1 h of exposure; it remained elevated throughout the 24-h incubation period shown by Western blot and ELISA. In addition, P. endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P < 0.05). IL-1alpha and P. endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

Lysophosphatidic acid stimulates epidermal growth factor-family ectodomain shedding and paracrine signaling from human lung fibroblasts.

PubMed

Shiomi, Tetsuya; Boudreault, Francis; Padem, Nurcicek; Higashiyama, Shigeki; Drazen, Jeffrey M; Tschumperlin, Daniel J

2011-01-01

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase-tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme-linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase-tagged heparin-binding epidermal growth factor (HB-EGF), amphiregulin, and transforming growth factor-a; non-transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB-EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal-regulated kinase 1/2, enhanced expression of mRNA for c-fos, HB-EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA-stimulated cells, and found enhanced EGFR and extracellular signal-regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF-family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells. © 2011 by the Wound Healing Society.

Electrical Stimulation Modulates High γ Activity and Human Memory Performance

PubMed Central

Berry, Brent M.; Miller, Laura R.; Khadjevand, Fatemeh; Ezzyat, Youssef; Wanda, Paul; Sperling, Michael R.; Lega, Bradley; Stead, S. Matt

2018-01-01

Direct electrical stimulation of the brain has emerged as a powerful treatment for multiple neurological diseases, and as a potential technique to enhance human cognition. Despite its application in a range of brain disorders, it remains unclear how stimulation of discrete brain areas affects memory performance and the underlying electrophysiological activities. Here, we investigated the effect of direct electrical stimulation in four brain regions known to support declarative memory: hippocampus (HP), parahippocampal region (PH) neocortex, prefrontal cortex (PF), and lateral temporal cortex (TC). Intracranial EEG recordings with stimulation were collected from 22 patients during performance of verbal memory tasks. We found that high γ (62–118 Hz) activity induced by word presentation was modulated by electrical stimulation. This modulatory effect was greatest for trials with “poor” memory encoding. The high γ modulation correlated with the behavioral effect of stimulation in a given brain region: it was negative, i.e., the induced high γ activity was decreased, in the regions where stimulation decreased memory performance, and positive in the lateral TC where memory enhancement was observed. Our results suggest that the effect of electrical stimulation on high γ activity induced by word presentation may be a useful biomarker for mapping memory networks and guiding therapeutic brain stimulation. PMID:29404403

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans.

PubMed

Osman, Narin; Grande-Allen, K Jane; Ballinger, Mandy L; Getachew, Robel; Marasco, Silvana; O'Brien, Kevin D; Little, Peter J

2013-01-01

Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-β1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factorβ1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-β1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-β1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease. Diseased human aortic valve leaflets demonstrated strong immunohistochemical staining for transforming growth factor-β1 and phosphorylated Smad2/3. In primary porcine aortic valve interstitial cells, Western blots showed that transforming growth factor-β1 stimulated phosphorylation in both the carboxy and linker regions of Smad2/3, which was inhibited by the transforming growth factor-β1 receptor inhibitor SB431542. Gel electrophoresis and size exclusion chromatography demonstrated that SB431542 decreased transforming growth factor-β1-mediated [(35)S]-sulfate incorporation into proteoglycans in a dose-dependent manner. Further, in proteoglycans derived from transforming growth factor-β1-treated valve interstitial cells, gel mobility shift assays demonstrated that inhibition of transforming growth factor-β1 receptor signaling resulted in decreased lipid binding. Classic transforming growth factor-β1 signaling is present in human aortic valves in vivo and contributes to the modification of proteoglycans expressed by valve interstitial cells in vitro. These findings suggest that transforming growth factor-β1 may promote increased low-density lipoprotein binding in the early phases of calcific aortic valve disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

PubMed Central

Hayashi, Yujiro; Asuzu, David T.; Gibbons, Simon J.; Aarsvold, Kirsten H.; Bardsley, Michael R.; Lomberk, Gwen A.; Mathison, Angela J.; Kendrick, Michael L.; Shen, K. Robert; Taguchi, Takahiro; Gupta, Anu; Rubin, Brian P.; Fletcher, Jonathan A.; Farrugia, Gianrico; Urrutia, Raul A.; Ordog, Tamas

2013-01-01

Stem cell factor (mouse: Kitl, human: KITLG) and insulin-like growth factor-1 (IGF1), acting via KIT and IGF1 receptor (IGF1R), respectively, are critical for the development and integrity of several tissues. Autocrine/paracrine KITLG-KIT and IGF1-IGF1R signaling are also activated in several cancers including gastrointestinal stromal tumors (GIST), the most common sarcoma. In murine gastric muscles, IGF1 promotes Kitl-dependent development of interstitial cells of Cajal (ICC), the non-neoplastic counterpart of GIST, suggesting cooperation between these pathways. Here, we report a novel mechanism linking IGF1-IGF1R and KITLG-KIT signaling in both normal and neoplastic cells. In murine gastric muscles, the microenvironment for ICC and GIST, human hepatic stellate cells (LX-2), a model for cancer niches, and GIST cells, IGF1 stimulated Kitl/KITLG protein and mRNA expression and promoter activity by activating several signaling pathways including AKT-mediated glycogen synthase kinase-3β inhibition (GSK3i). GSK3i alone also stimulated Kitl/KITLG expression without activating mitogenic pathways. Both IGF1 and GSK3i induced chromatin-level changes favoring transcriptional activation at the Kitl promoter including increased histone H3/H4 acetylation and H3 lysine (K) 4 methylation, reduced H3K9 and H3K27 methylation and reduced occupancy by the H3K27 methyltransferase EZH2. By pharmacological or RNA interference-mediated inhibition of chromatin modifiers we demonstrated that these changes have the predicted impact on KITLG expression. KITLG knock-down and immunoneutralization inhibited the proliferation of GIST cells expressing wild-type KIT, signifying oncogenic autocrine/paracrine KITLG-KIT signaling. We conclude that membrane-to-nucleus signaling involving GSK3i establishes a previously unrecognized link between the IGF1-IGF1R and KITLG-KIT pathways, which is active in both physiologic and oncogenic contexts and can be exploited for therapeutic purposes. PMID:24116170

IRAK4 kinase activity controls Toll-like receptor-induced inflammation through the transcription factor IRF5 in primary human monocytes.

PubMed

Cushing, Leah; Winkler, Aaron; Jelinsky, Scott A; Lee, Katherine; Korver, Wouter; Hawtin, Rachael; Rao, Vikram R; Fleming, Margaret; Lin, Lih-Ling

2017-11-10

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate immune signaling by Toll-like receptors (TLRs), and loss of IRAK4 activity in mice and humans increases susceptibility to bacterial infections and causes defects in TLR and IL1 ligand sensing. However, the mechanism by which IRAK4 activity regulates the production of downstream inflammatory cytokines is unclear. Using transcriptomic and biochemical analyses of human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the activation of interferon regulatory factor 5 (IRF5), a transcription factor implicated in the pathogenesis of multiple autoimmune diseases. Following TLR7/8 stimulation by its agonist R848, chemical inhibition of IRAK4 abolished IRF5 translocation to the nucleus and thus prevented IRF5 binding to and activation of the promoters of inflammatory cytokines in human monocytes. We also found that IKKβ, an upstream IRF5 activator, is phosphorylated in response to the agonist-induced TLR signaling. Of note, IRAK4 inhibition blocked IKKβ phosphorylation but did not block the nuclear translocation of NFκB, which was surprising, given the canonical role of IKKβ in phosphorylating IκB to allow NFκB activation. Moreover, pharmacological inhibition of either IKKβ or the serine/threonine protein kinase TAK1 in monocytes blocked TLR-induced cytokine production and IRF5 translocation to the nucleus, but not nuclear translocation of NFκB. Taken together, our data suggest a mechanism by which IRAK4 activity regulates TAK1 and IKKβ activation, leading to the nuclear translocation of IRF5 and induction of inflammatory cytokines in human monocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

PubMed Central

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-01-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells. PMID:12133007

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

PubMed

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-11-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

Effects of cranberry components on IL-1β-stimulated production of IL-6, IL-8 and VEGF by human TMJ synovial fibroblasts.

PubMed

Tipton, David A; Christian, James; Blumer, Adam

2016-08-01

Osteoarthritis (OA) in the TMJ is characterized by deterioration of articular cartilage and secondary inflammatory changes. Interleukin-1β (IL-1β) stimulates IL-6, IL-8, and vascular endothelial growth factor (VEGF) in synovial fluid of TMJ with internal derangement and bony changes. The cranberry (Vaccinium macrocarpon) contains polyphenolic compounds that inhibit production of pro-inflammatory molecules by gingival cells in response to several stimulators. This study examined effects of cranberry components on IL-1β-stimulated IL-6, IL-8, and VEGF production by human TMJ synovial fibroblast-like cells. Cranberry high molecular weight non-dialyzable material (NDM) was derived from cranberry juice. Human TMJ synovial fibroblast-like cells from joints with degenerative OA and an ankylosed TMJ without degeneration were incubated with IL-1β (0.001-1nM)±NDM (25-250μg/ml) (2h preincubation). Viability was assessed via activity of a mitochondrial enzyme. IL-6, IL-8, and VEGF in culture supernatants were measured by ELISA; NF-κB and AP-1 transcription factors were measured in nuclear extracts via binding to specific oligonucleotides. ANOVA and Scheffe's F procedure for post hoc comparisons. NDM did not affect cell viability but inhibited IL-1β stimulated IL-6, IL-8, and VEGF production in all cell lines (p<0.05). NDM partially reduced nuclear levels of NF-κB and AP-1 (p<0.04), depending upon cell line and time of exposure to IL-1β+NDM. Cranberry NDM inhibition of IL-1β-stimulated IL- 6, IL-8, and VEGF production by TMJ synovial fibroblast-like cells suggests that cranberry components may be useful as a host modulatory therapeutic agent to prevent or treat inflammatory arthropathies of the TMJ. Copyright © 2016 Elsevier Ltd. All rights reserved.

Co-incubation of PMN and CaCo-2 cells modulates inflammatory potential.

PubMed

Schaefer, M B; Schaefer, C A; Hecker, M; Morty, R E; Witzenrath, M; Seeger, W; Mayer, K

2017-05-20

Polymorphonuclear granulocytes (PMN) are activated in inflammatory reactions. Intestinal epithelial cells are relevant for maintaining the intestinal barrier. We examined interactions of PMN and intestinal epithelial cell-like CaCo-2 cells to elucidate their regulation of inflammatory signalling and the impact of cyclooxygenase (COX), nitric oxide (NO) and platelet-activating factor (PAF). Human PMN and CaCo-2 cells, separately and in co-incubation, were stimulated with the calcium ionophore A23187 or with N-Formyl-methionyl-leucyl-phenylalanin (fMLP) that activates PMN only. Human neutrophil elastase (HNE) and respiratory Burst were measured. To evaluate the modulation of inflammatory crosstalk we applied inhibitors of COX (acetyl salicylic acid; ASA), NO-synthase (N-monomethyl-L-arginin; L-NMMA), and the PAF-receptor (WEB2086). Unstimulated, co-incubation of CaCo-2 cells and PMN led to significantly reduced Burst and elevated HNE as compared to PMN. After stimulation with A23187, co-incubation resulted in an inhibition of Burst and HNE. Using fMLP co-incubation failed to modulate Burst but increased HNE. Without stimulation, all three inhibitors abolished the effect of co-incubation on Burst but did not change HNE.  ASA partly prevented modulation of Burst L-NMMA and WEB2086 did not change Burst but abolished mitigation of HNE. Without stimulation, co-incubation reduced Burst and elevated HNE. Activation of PMN and CaCo-2 cells by fMLP as compared to A23187 resulted in a completely different pattern of Burst and HNE, possibly due to single vs. dual cell activation. Anti-inflammatory effect of co-incubation might in part be due to due to COX-signalling governing Burst whereas NO- and PAF-dependent signalling seemed to control HNE release.

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

PubMed

Leitner, Gerda C; Faschingbauer, Martin; Wenda, Sabine; Weigel, Günter; Fischer, Gottfried

2014-12-01

The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors. © 2014 AABB.

Cysteine, histidine and glycine exhibit anti-inflammatory effects in human coronary arterial endothelial cells.

PubMed

Hasegawa, S; Ichiyama, T; Sonaka, I; Ohsaki, A; Okada, S; Wakiguchi, H; Kudo, K; Kittaka, S; Hara, M; Furukawa, S

2012-02-01

The activation of nuclear factor-kappa B (NF-κB) in vascular endothelial cells may be involved in vascular pathogeneses such as vasculitis or atherosclerosis. Recently, it has been reported that some amino acids exhibit anti-inflammatory effects. We investigated the inhibitory effects of a panel of amino acids on cytokine production or expression of adhesion molecules that are involved in inflammatory diseases in various cell types. The activation of NF-κB was determined in human coronary arterial endothelial cells (HCAECs) because NF-κB modulates the production of many cytokines and the expression of adhesion molecules. We examined the inhibitory effects of the amino acids cysteine, histidine and glycine on the induction of NF-κB activation, expression of CD62E (E-selectin) and the production of interleukin (IL)-6 in HCAECs stimulated with tumour necrosis factor (TNF)-α. Cysteine, histidine and glycine significantly reduced NF-κB activation and inhibitor κBα (IκBα) degradation in HCAECs stimulated with TNF-α. Additionally, all the amino acids inhibited the expression of E-selectin and the production of IL-6 in HCAECs, and the effects of cysteine were the most significant. Our results show that glycine, histidine and cysteine can inhibit NF-κB activation, IκBα degradation, CD62E expression and IL-6 production in HCAECs, suggesting that these amino acids may exhibit anti-inflammatory effects during endothelial inflammation. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

Network connectivity and individual responses to brain stimulation in the human motor system.

PubMed

Cárdenas-Morales, Lizbeth; Volz, Lukas J; Michely, Jochen; Rehme, Anne K; Pool, Eva-Maria; Nettekoven, Charlotte; Eickhoff, Simon B; Fink, Gereon R; Grefkes, Christian

2014-07-01

The mechanisms driving cortical plasticity in response to brain stimulation are still incompletely understood. We here explored whether neural activity and connectivity in the motor system relate to the magnitude of cortical plasticity induced by repetitive transcranial magnetic stimulation (rTMS). Twelve right-handed volunteers underwent functional magnetic resonance imaging during rest and while performing a simple hand motor task. Resting-state functional connectivity, task-induced activation, and task-related effective connectivity were assessed for a network of key motor areas. We then investigated the effects of intermittent theta-burst stimulation (iTBS) on motor-evoked potentials (MEP) for up to 25 min after stimulation over left primary motor cortex (M1) or parieto-occipital vertex (for control). ITBS-induced increases in MEP amplitudes correlated negatively with movement-related fMRI activity in left M1. Control iTBS had no effect on M1 excitability. Subjects with better response to M1-iTBS featured stronger preinterventional effective connectivity between left premotor areas and left M1. In contrast, resting-state connectivity did not predict iTBS aftereffects. Plasticity-related changes in M1 following brain stimulation seem to depend not only on local factors but also on interconnected brain regions. Predominantly activity-dependent properties of the cortical motor system are indicative of excitability changes following induction of cortical plasticity with rTMS. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Polysaccharide from black currant (Ribes nigrum L.) stimulates dendritic cells through TLR4 signaling.

PubMed

Ashigai, Hiroshi; Komano, Yuta; Wang, Guanying; Kawachi, Yasuji; Sunaga, Kazuko; Yamamoto, Reiko; Takata, Ryoji; Miyake, Mika; Yanai, Takaaki

2017-01-01

Black currant ( Ribes nigrum ) has various beneficial properties for human health. In particular, polysaccharide from black currant was found to be an immunostimulating food ingredient and was reported to have antitumor activity in a mouse model. We named it cassis polysaccharide (CAPS). In a previous study, CAPS administration caused tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production in vitro and in vivo , but the immunological mechanism of CAPS was not demonstrated. In this study, we revealed the CAPS immunostimulating mechanism in vitro . First, we found that CAPS activated dendritic cells (DCs). Second, we investigated whether it depends on Toll-like receptor 4 (TLR4) and myeloid differentiation primary response (Myd). We concluded that CAPS stimulates DCs through Myd88 depending TLR4 signaling and activates Th1-type cytokine release.

Rough titanium alloys regulate osteoblast production of angiogenic factors.

PubMed

Olivares-Navarrete, Rene; Hyzy, Sharon L; Gittens, Rolando A; Schneider, Jennifer M; Haithcock, David A; Ullrich, Peter F; Slosar, Paul J; Schwartz, Zvi; Boyan, Barbara D

2013-11-01

Polyether-ether-ketone (PEEK) and titanium-aluminum-vanadium (titanium alloy) are used frequently in lumbar spine interbody fusion. Osteoblasts cultured on microstructured titanium generate an environment characterized by increased angiogenic factors and factors that inhibit osteoclast activity mediated by integrin α2β1 signaling. It is not known if this is also true of osteoblasts on titanium alloy or PEEK. The purpose of this study was to determine if osteoblasts generate an environment that supports angiogenesis and reduces osteoclastic activity when grown on smooth titanium alloy, rough titanium alloy, or PEEK. This in vitro study compared angiogenic factor production and integrin gene expression of human osteoblast-like MG63 cells cultured on PEEK or titanium-aluminum-vanadium (titanium alloy). MG63 cells were grown on PEEK, smooth titanium alloy, or rough titanium alloy. Osteogenic microenvironment was characterized by secretion of osteoprotegerin and transforming growth factor beta-1 (TGF-β1), which inhibit osteoclast activity and angiogenic factors including vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2), and angiopoietin-1 (ANG-1). Expression of integrins, transmembrane extracellular matrix recognition proteins, was measured by real-time polymerase chain reaction. Culture on titanium alloy stimulated osteoprotegerin, TGF-β1, VEGF-A, FGF-2, and angiopoietin-1 production, and levels were greater on rough titanium alloy than on smooth titanium alloy. All factors measured were significantly lower on PEEK than on smooth or rough titanium alloy. Culture on titanium alloy stimulated expression of messenger RNA for integrins that recognize Type I collagen in comparison with PEEK. Rough titanium alloy stimulated cells to create an osteogenic-angiogenic microenvironment. The osteogenic-angiogenic responses to titanium alloy were greater than PEEK and greater on rough titanium alloy than on smooth titanium alloy. Surface features regulated expression of integrins important in collagen recognition. These factors may increase bone formation, enhance integration, and improve implant stability in interbody spinal fusions. Copyright © 2013 Elsevier Inc. All rights reserved.

Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Montgomery, K.F.; Tarr, P.I.; Bomsztyk, K.

1991-08-01

Leukocyte adherence to endothelium is in part mediated by the transient expression of endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial surfaces stimulated by tumor necrosis factor {alpha} (TNF), interleukin (IL) 1, or bacterial lipopolysaccharide (LPS). The intracellular factors controlling induction of ELAM-1 mRNA and protein are unknown. In nuclear runoff experiments with cultured human umbilical vein endothelial cells (HUVEC), the authors demonstrate that transcriptional activation of the ELAM-1 gene occurs following stimulation with TNF. Sequence analysis of the 5{prime} flanking region of the ELAM-1 gene reveals consensus DNA-binding sequences for two known transcription factors, NF-{kappa}B and AP-1. Gel mobility shiftmore » assays demonstrate that TNF, IL-1, or LPS induces activation of NF-{kappa}B-like DNA binding activity in HUVEC. Phorbol 12-myristate 13-acetate, a known activator of protein kinase C (PKC), weakly induces NF-{kappa}B-like activity, ELAM-1 mRNA, and ELAM-1 surface expression in HUVEC. However, TNF, IL-1, and LPS do not activate PKC in HUVEC at doses that strongly induce NF-{kappa}B-like protein activation and ELAM-1 gene expression. PKC blockade with H7 does not inhibit activation of these NF-kB-like proteins but does inhibit ELAM-1 gene transcription. They conclude that PKC-independent activation of NF-{kappa}B in HUVEC with TNF, IL-1, or LPS is associated with, but not sufficient for, activation of ELAM-1 gene transcription.« less

Endothelial atheroprotective and anti-inflammatory mechanisms.

PubMed

Berk, B C; Abe, J I; Min, W; Surapisitchat, J; Yan, C

2001-12-01

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF-mediated EC activation. EC apoptosis is likely important in the process termed "plaque erosion" that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide-dependent protective pathways that involve enzymes PI3-kinase and Akt. Conversely, our laboratory has identified nitric oxide-independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen-activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF-mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF-stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre-exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen-activated protein kinase kinase inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Flow-mediated inhibition of JNK was unaffected by 0.1 mM L-nitroarginine, 100 pM 8-bromo-cyclic GMP, or 100 microM 8-bromo-cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow-mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Urothelium muscarinic activation phosphorylates CBSSer227 via cGMP/PKG pathway causing human bladder relaxation through H2S production

PubMed Central

d’Emmanuele di Villa Bianca, Roberta; Mitidieri, Emma; Fusco, Ferdinando; Russo, Annapina; Pagliara, Valentina; Tramontano, Teresa; Donnarumma, Erminia; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

2016-01-01

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser227 following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders. PMID:27509878

Urothelium muscarinic activation phosphorylates CBS(Ser227) via cGMP/PKG pathway causing human bladder relaxation through H2S production.

PubMed

d'Emmanuele di Villa Bianca, Roberta; Mitidieri, Emma; Fusco, Ferdinando; Russo, Annapina; Pagliara, Valentina; Tramontano, Teresa; Donnarumma, Erminia; Mirone, Vincenzo; Cirino, Giuseppe; Russo, Giulia; Sorrentino, Raffaella

2016-08-11

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser(227) following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders.

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

PubMed

Stockis, Julie; Colau, Didier; Coulie, Pierre G; Lucas, Sophie

2009-12-01

Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.

BAFF, a Novel Ligand of the Tumor Necrosis Factor Family, Stimulates B Cell Growth

PubMed Central

Schneider, Pascal; MacKay, Fabienne; Steiner, Véronique; Hofmann, Kay; Bodmer, Jean-Luc; Holler, Nils; Ambrose, Christine; Lawton, Pornsri; Bixler, Sarah; Acha-Orbea, Hans; Valmori, Danila; Romero, Pedro; Werner-Favre, Christiane; Zubler, Rudolph H.; Browning, Jeffrey L.; Tschopp, Jürg

1999-01-01

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M–stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center–like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function. PMID:10359578

Mode of action of the immunostimulatory effect of collagen from jellyfish.

PubMed

Nishimoto, Sogo; Goto, Yoko; Morishige, Hitoshi; Shiraishi, Ryusuke; Doi, Mikiharu; Akiyama, Koichi; Yamauchi, Satoshi; Sugahara, Takuya

2008-11-01

We have previously demonstrated that collagen from jellyfish simulated immunoglobulin and cytokine production by human-human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). The mode of action of the collagen as an immunostimulatory factor was investigated. The expression levels of immunoglobulin mRNAs in HB4C5 cells, and those of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta in hPBL were up-regulated by jellyfish collagen. In addition, this collagen activated IgM production by transcription-suppressed HB4C5 cells that had been treated with actinomycin D. This collagen also enhanced IgM production by translation-suppressed HB4C5 cells that had been treated with sodium fluoride, but was ineffective in accelerating IgM production by HB4C5 cells treated with cycloheximide. Moreover, the intracellular IgM level in HB4C5 cells treated with the post-translation inhibitor, monensin, was increased by this collagen. These results suggest that collagen from jellyfish stimulated not only the transcription activity, but also the translation activity for enhanced immunoglobulin and cytokine production.

Bromelain Treatment Decreases Secretion of Pro-Inflammatory Cytokines and Chemokines by Colon Biopsies In Vitro

PubMed Central

Onken, Jane E.; Greer, Paula K.; Calingaert, Brian; Hale, Laura P.

2008-01-01

Oral bromelain has been anecdotally reported to decrease inflammation in ulcerative colitis (UC). Proteolytically active bromelain is known to decrease expression of mRNAs encoding pro-inflammatory cytokines by human leukocytes in vitro. To assess the effect of bromelain on mucosal secretion of cytokines in inflammatory bowel disease (IBD), endoscopic colon biopsies from patients with UC, Crohn’s disease (CD), and non-IBD controls were treated in vitro with bromelain or media, then cultured. Secretion of pro-inflammatory cytokines and chemokines was measured. Significant increases in granulocyte colony stimulating factor (G-CSF), interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) were detected in the media from actively inflamed areas in UC and CD as compared with non-inflamed IBD tissue and non-IBD controls. In vitro bromelain treatment decreased secretion of G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), IFN-γ, CCL4/macrophage inhibitory protein (MIP)-1β, and TNF by inflamed tissue in IBD. Bromelain may be a novel therapy for IBD. PMID:18160345

Adaptogens Stimulate Neuropeptide Y and Hsp72 Expression and Release in Neuroglia Cells

PubMed Central

Panossian, Alexander; Wikman, Georg; Kaur, Punit; Asea, Alexzander

2011-01-01

The beneficial stress–protective effect of adaptogens is related to the regulation of homeostasis via mechanisms of action associated with the hypothalamic–pituitary–adrenal axis and the regulation of key mediators of the stress response, such as molecular chaperones, stress-activated c-Jun N-terminal protein kinase, forkhead box O transcription factor, cortisol, and nitric oxide (NO). However, it still remains unclear what the primary upstream targets are in response to stimulation by adaptogens. The present study addresses this gap in our knowledge and suggests that an important target for adaptogen mediated stress–protective effector functions is the stress hormone neuropeptide Y (NPY). We demonstrated that ADAPT-232, a fixed combination of adaptogens Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5, and its active constituent salidroside, stimulated the expression of NPY and 72 kDa heat shock protein (Hsp72) in isolated human neuroglia cells. The central role of NPY was validated in experiments in which pre-treatment of human neuroglia cells with NPY-siRNA and HSF1-siRNA resulted in the significant suppression of ADAPT-232-induced NPY and Hsp72 release. Taken together our studies suggest that the stimulation and release of the stress hormones, NPY and Hsp72, into systemic circulation is an innate defense response against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress. PMID:22347152

Adaptogens stimulate neuropeptide y and hsp72 expression and release in neuroglia cells.

PubMed

Panossian, Alexander; Wikman, Georg; Kaur, Punit; Asea, Alexzander

2012-01-01

The beneficial stress-protective effect of adaptogens is related to the regulation of homeostasis via mechanisms of action associated with the hypothalamic-pituitary-adrenal axis and the regulation of key mediators of the stress response, such as molecular chaperones, stress-activated c-Jun N-terminal protein kinase, forkhead box O transcription factor, cortisol, and nitric oxide (NO). However, it still remains unclear what the primary upstream targets are in response to stimulation by adaptogens. The present study addresses this gap in our knowledge and suggests that an important target for adaptogen mediated stress-protective effector functions is the stress hormone neuropeptide Y (NPY). We demonstrated that ADAPT-232, a fixed combination of adaptogens Eleutherococcus senticosus root extract, Schisandra chinensis berry extract, Rhodiola rosea root extract SHR-5, and its active constituent salidroside, stimulated the expression of NPY and 72 kDa heat shock protein (Hsp72) in isolated human neuroglia cells. The central role of NPY was validated in experiments in which pre-treatment of human neuroglia cells with NPY-siRNA and HSF1-siRNA resulted in the significant suppression of ADAPT-232-induced NPY and Hsp72 release. Taken together our studies suggest that the stimulation and release of the stress hormones, NPY and Hsp72, into systemic circulation is an innate defense response against mild stressors (ADAPT-232), which increase tolerance and adaptation to stress.

Bm-TFF2, a toad trefoil factor, promotes cell migration, survival and wound healing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yong; Graduate School of Chinese Academy of Sciences, Beijing 100049; Yu, Guoyu

2010-07-30

Research highlights: {yields} Bm-TFF2 binds to epithelial cells and induces cell migration and wound healing. {yields} Bm-TFF2 suppresses cell apoptosis. {yields} Bm-TFF2 has no effect on cell proliferation. -- Abstract: Toad skin is naked and continually confronted by various injurious factors. Constant skin renewal and repairs occur frequently. However, the mechanisms of the renewal and repair have not clearly elucidated. In our previous work, a trefoil factor (TFF), Bm-TFF2, has been purified from the Bombina maxima skin and characterized as a platelet agonist. The mRNA of TFFs in toad skin was up-regulated greatly during the metamorphosis, indicating a pivotal rolemore » of TFFs in amphibian skin. Here, we presented the effects of Bm-TFF2 on the cell migration, apoptosis and proliferation. Bm-TFF2 bound to epithelial cells and showed strong cell motility activity. At the concentrations of 1-100 nM, Bm-TFF2-induced migration of human epithelial AGS and HT-29 cells, and rat intestinal epithelial IEC-6 cell lines. The in vitro wound healing assay also verified the activity of Bm-TFF2. Bm-TFF2 could also inhibit cell apoptosis induced by ceramide and sodium butyrate. The cell migration-promoting activity was abolished by MEK1 inhibitors, U0126 and PD98059, suggesting that ERK1/2 activation is crucial for Bm-TFF2 to stimulate cell migration. Taken together, Bm-TFF2 promoted wound healing by stimulating cell migration via MAPK pathway and preventing cell apoptosis. The potent biological activity of Bm-TFF2 makes it a useful molecular tool for further studies of structure-function relationship of the related human TFFs.« less

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization.

PubMed

Vanz, Ana Ls; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

2008-04-04

Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

PubMed Central

Vanz, Ana LS; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

2008-01-01

Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community. PMID:18394164

Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

PubMed

Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

2016-01-01

Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of Immune Responses Mediated by Listeria-Stimulated Human Dendritic Cells: Implications for Cancer Vaccine Therapy

DTIC Science & Technology

2015-09-01

Award Number: W81XWH-11-1-0384 TITLE: Evaluation of Immune Responses Mediated by Listeria-Stimulated Human Dendritic Cells : Implications for...Immune Responses Mediated by Listeria-Stimulated Human Dendritic Cells : Implications for Cancer Vaccine Therapy 5b. GRANT NUMBER CA100463 5c...Listeria monocytogenes (Lm) on human dendritic cells (DCs) to optimize Lm-based DC cancer vaccines. The project aims are: 1) Compare the activation and

Hyaluronan inhibits prostaglandin E2 production via CD44 in U937 human macrophages.

PubMed

Yasuda, Tadashi

2010-03-01

Prostaglandin E(2) (PGE(2)) is one of the key mediators of inflammation in affected joints of rheumatoid arthritis (RA). Intra-articular injection of high molecular weight hyaluronan (HA) into RA knee joints relieves arthritic pain. Although HA has been shown to inhibit PGE(2) production in cytokine-stimulated synovial fibroblasts, it remains unclear how HA suppresses PGE(2) production in activated cells. Furthermore, HA effect on macrophages has rarely been investigated in spite of their contribution to RA joint pathology. This study was aimed to investigate the inhibitory mechanism of HA on lipopolysaccharide (LPS)-stimulated PGE(2) production in U937 human macrophages. Stimulation of U937 macrophages with LPS enhanced PGE(2) production in association with increased protein levels of cyclooxygenase-2 (COX-2). Pretreatment with HA of 2,700 kDa resulted in suppression of the LPS-mediated induction of COX-2, leading to a decrease in PGE(2) production. Likewise, the LPS-stimulated PGE(2) production was inhibited by the pretreatment with a specific COX2 inhibitor, NS-398, or a specific inhibitor of nuclear factor (NF)-kappaB, BAY11-7085. HA also decreased the degree of phosphorylation and nuclear translocation of NF-kappaB enhanced by LPS. Fluorescence cytochemistry demonstrated that HA bound to CD44, the principal HA receptor, on U937 macrophages. Anti-CD44 antibody reversed the inhibitory effects of HA on the LPS-mediated increase in PGE(2) production, COX-2 induction, and activation of NF-kappaB. These results indicate that HA suppresses the LPS-stimulated PGE(2) production via CD44 through down-regulation of NF-kappaB. Administration of HA into RA joints may decrease PGE(2) production by activated macrophages, which could result in improvement of arthritic pain.

Comparison of electric field strength and spatial distribution of electroconvulsive therapy and magnetic seizure therapy in a realistic human head model

PubMed Central

Lee, Won Hee; Lisanby, Sarah H.; Laine, Andrew F.; Peterchev, Angel V.

2017-01-01

Background This study examines the strength and spatial distribution of the electric field induced in the brain by electroconvulsive therapy (ECT) and magnetic seizure therapy (MST). Methods The electric field induced by standard (bilateral, right unilateral, and bifrontal) and experimental (focal electrically administered seizure therapy and frontomedial) ECT electrode configurations as well as a circular MST coil configuration was simulated in an anatomically realistic finite element model of the human head. Maps of the electric field strength relative to an estimated neural activation threshold were used to evaluate the stimulation strength and focality in specific brain regions of interest for these ECT and MST paradigms and various stimulus current amplitudes. Results The standard ECT configurations and current amplitude of 800–900 mA produced the strongest overall stimulation with median of 1.8–2.9 times neural activation threshold and more than 94% of the brain volume stimulated at suprathreshold level. All standard ECT electrode placements exposed the hippocampi to suprathreshold electric field, although there were differences across modalities with bilateral and right unilateral producing respectively the strongest and weakest hippocampal stimulation. MST stimulation is up to 9 times weaker compared to conventional ECT, resulting in direct activation of only 21% of the brain. Reducing the stimulus current amplitude can make ECT as focal as MST. Conclusions The relative differences in electric field strength may be a contributing factor for the cognitive sparing observed with right unilateral compared to bilateral ECT, and MST compared to right unilateral ECT. These simulations could help understand the mechanisms of seizure therapies and develop interventions with superior risk/benefit ratio. PMID:27318858

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

PubMed

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Asbestos and erionite prime and activate the NLRP3 inflammasome that stimulates autocrine cytokine release in human mesothelial cells

PubMed Central

2013-01-01

Background Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear. Results We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model. Conclusions These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis. PMID:23937860

Withaferin A inhibits tumor necrosis factor-alpha-induced expression of cell adhesion molecules by inactivation of Akt and NF-kappaB in human pulmonary epithelial cells.

PubMed

Oh, Jung Hwa; Kwon, Taeg Kyu

2009-05-01

We here investigated the functional effect of withaferin A on airway inflammation and its action mechanism. Withaferin A inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human lung epithelial A549 cells stimulated with tumor necrosis factor-alpha (TNF-alpha), resulting in the suppression of leukocyte adhesion to lung epithelial A549 cells. In addition, withaferin A inhibited TNF-alpha-induced expression of adhesion molecules (ICAM-1 and VCAM-1) protein and mRNA in a dose-dependent manner. Withaferin A prevented DNA binding activity of nuclear factor-kappaB (NF-kappaB) and nuclear translocation of NF-kappaB. It also inhibited phosphorylation of Akt and extracellular signal-regulated kinase (ERK), which are upstream in the regulation of adhesion molecules by TNF-alpha. Furthermore, withaferin A inhibited U937 monocyte adhesion to A549 cells stimulated by TNF-alpha, suggesting that it may inhibit the binding of these cells by regulating the expression of critical adhesion molecules by TNF-alpha. Taken together, these results suggest that withaferin A inhibits cell adhesion through inhibition of ICAM-1 and VCAM-1 expression, at least in part, by blocking Akt and down-regulating NF-kappaB activity.

Can the human lumbar posterior columns be stimulated by transcutaneous spinal cord stimulation? A modeling study

PubMed Central

Danner, Simon M.; Hofstoetter, Ursula S.; Ladenbauer, Josef; Rattay, Frank; Minassian, Karen

2014-01-01

Stimulation of different spinal cord segments in humans is a widely developed clinical practice for modification of pain, altered sensation and movement. The human lumbar cord has become a target for modification of motor control by epidural and more recently by transcutaneous spinal cord stimulation. Posterior columns of the lumbar spinal cord represent a vertical system of axons and when activated can add other inputs to the motor control of the spinal cord than stimulated posterior roots. We used a detailed three-dimensional volume conductor model of the torso and the McIntyre-Richard-Grill axon model to calculate the thresholds of axons within the posterior columns in response to transcutaneous lumbar spinal cord stimulation. Superficially located large diameter posterior column fibers with multiple collaterals have a threshold of 45.4 V, three times higher than posterior root fibers (14.1 V). With the stimulation strength needed to activate posterior column axons, posterior root fibers of large and small diameters as well as anterior root fibers are co-activated. The reported results inform on these threshold differences, when stimulation is applied to the posterior structures of the lumbar cord at intensities above the threshold of large-diameter posterior root fibers. PMID:21401670

Regulation of B7.1 costimulatory molecule is mediated by the IFN regulatory factor-7 through the activation of JNK in lipopolysaccharide-stimulated human monocytic cells.

PubMed

Lim, Wilfred; Gee, Katrina; Mishra, Sasmita; Kumar, Ashok

2005-11-01

The engagement of CD28 or CTLA-4 with B7.1 provides the essential second costimulatory signal that regulates the development of immune responses, including T cell activation, differentiation, and induction of peripheral tolerance. The signaling molecules and the transcription factors involved in B7.1 regulation are poorly understood. In this study we investigated the role of MAPKs in the regulation of LPS-induced B7.1 expression in human monocytes and the promonocytic THP-1 cells. Our results show that LPS-induced B7.1 expression in monocytic cells did not involve the activation of either p38 or ERKs. Using the JNK-specific inhibitor SP600125, small interfering RNAs specific for JNK1 and JNK2, and agents such as dexamethasone that inhibit JNK activation, we determined that LPS-induced B7.1 expression was regulated by JNK MAPK in both monocytes and THP-1 cells. In addition, we identified a distinct B7.1-responsive element corresponding to the IFN regulatory factor-7 (IRF-7) binding site in the B7.1 promoter responsible for the regulation of LPS-induced B7.1 transcription. Furthermore, SP600125 and dexamethasone inhibited LPS-induced IRF-7 activity. Taken together, these results suggest that LPS-induced B7.1 transcription in human monocytic cells may be regulated by JNK-mediated activation of the IRF-7 transcription factor.

Mechanical Stimulation (Pulsed Electromagnetic Fields “PEMF” and Extracorporeal Shock Wave Therapy “ESWT”) and Tendon Regeneration: A Possible Alternative

PubMed Central

Rosso, Federica; Bonasia, Davide E.; Marmotti, Antonio; Cottino, Umberto; Rossi, Roberto

2015-01-01

The pathogenesis of tendon degeneration and tendinopathy is still partially unclear. However, an active role of metalloproteinases (MMP), growth factors, such as vascular endothelial growth factor (VEGF) and a crucial role of inflammatory elements and cytokines was demonstrated. Mechanical stimulation may play a role in regulation of inflammation. In vitro studies demonstrated that both pulsed electromagnetic fields (PEMF) and extracorporeal shock wave therapy (ESWT) increased the expression of pro-inflammatory cytokine such as interleukin (IL-6 and IL-10). Moreover, ESWT increases the expression of growth factors, such as transforming growth factor β(TGF-β), (VEGF), and insulin-like growth factor 1 (IGF1), as well as the synthesis of collagen I fibers. These pre-clinical results, in association with several clinical studies, suggest a potential effectiveness of ESWT for tendinopathy treatment. Recently PEMF gained popularity as adjuvant for fracture healing and bone regeneration. Similarly to ESWT, the mechanical stimulation obtained using PEMFs may play a role for treatment of tendinopathy and for tendon regeneration, increasing in vitro TGF-β production, as well as scleraxis and collagen I gene expression. In this manuscript the rational of mechanical stimulations and the clinical studies on the efficacy of extracorporeal shock wave (ESW) and PEMF will be discussed. However, no clear evidence of a clinical value of ESW and PEMF has been found in literature with regards to the treatment of tendinopathy in human, so further clinical trials are needed to confirm the promising hypotheses concerning the effectiveness of ESWT and PEMF mechanical stimulation. PMID:26617513

A functional magnetic resonance imaging study of human brain in pain-related areas induced by electrical stimulation with different intensities.

PubMed

Yuan, Wang; Ming, Zhang; Rana, Netra; Hai, Liu; Chen-wang, Jin; Shao-hui, Ma

2010-01-01

Pain-related studies have mainly been performed through traditional methods, which lack the rigorous analysis of anatomical locations. Functional magnetic resonance imaging (fMRI) is a noninvasive method detecting neural activity, and has the ability to precisely locate related activations in vivo. Moreover, few studies have used painful stimulation of changed intensity to investigate relevant functioning nuclei in the human brain. This study mainly focused on the pain-related activations induced by electrical stimulation with different intensities using fMRI. Furthermore, the electrophysiological characteristics of different pain-susceptible-neurons were analyzed to construct the pain modulatory network, which was corresponding to painful stimulus of changed intensity. Twelve volunteers underwent functional scanning receiving different electrical stimulation. The data were collected and analyzed to generate the corresponding functional activation maps and response time curves related to pain. The common activations were mainly located in several specific regions, including the secondary somatosensory cortex (SII), insula, anterior cingulate cortex (ACC), thalamus, and other cerebral regions. Moreover, innocuous electrical stimulation primarily activated the lateral portions of SII and thalamus, as well as the posterior insula, anterior ACC, whereas noxious electrical stimulation primarily activated the medial portions of SII and thalamus, as well as the anterior insula, the posterior ACC, with larger extensions and greater intensities. Several specified cerebral regions displayed different response patterns during electrical stimulation by means of fMRI, which implied that the corresponding pain-susceptible-neurons might process specific aspects of pain. Elucidation of functions on pain-related regions will help to understand the delicate pain modulation of human brain.

Selective binding and oligomerization of the murine granulocyte colony-stimulating factor receptor by a low molecular weight, nonpeptidyl ligand.

PubMed

Doyle, Michael L; Tian, Shin-Shay; Miller, Stephen G; Kessler, Linda; Baker, Audrey E; Brigham-Burke, Michael R; Dillon, Susan B; Duffy, Kevin J; Keenan, Richard M; Lehr, Ruth; Rosen, Jon; Schneeweis, Lumelle A; Trill, John; Young, Peter R; Luengo, Juan I; Lamb, Peter

2003-03-14

Granulocyte colony-stimulating factor regulates neutrophil production by binding to a specific receptor, the granulocyte colony-stimulating factor receptor, expressed on cells of the granulocytic lineage. Recombinant forms of granulocyte colony-stimulating factor are used clinically to treat neutropenias. As part of an effort to develop granulocyte colony-stimulating factor mimics with the potential for oral bioavailability, we previously identified a nonpeptidyl small molecule (SB-247464) that selectively activates murine granulocyte colony-stimulating factor signal transduction pathways and promotes neutrophil formation in vivo. To elucidate the mechanism of action of SB-247464, a series of cell-based and biochemical assays were performed. The activity of SB-247464 is strictly dependent on the presence of zinc ions. Titration microcalorimetry experiments using a soluble murine granulocyte colony-stimulating factor receptor construct show that SB-247464 binds to the extracellular domain of the receptor in a zinc ion-dependent manner. Analytical ultracentrifugation studies demonstrate that SB-247464 induces self-association of the N-terminal three-domain fragment in a manner that is consistent with dimerization. SB-247464 induces internalization of granulocyte colony-stimulating factor receptor on intact cells, consistent with a mechanism involving receptor oligomerization. These data show that small nonpeptidyl compounds are capable of selectively binding and inducing productive oligomerization of cytokine receptors.

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

PubMed

Park, Hyun-Jeong; Choi, Bum-Chae; Song, Sang-Jin; Lee, Dong-Sik; Roh, Jaesook; Chun, Sang-Young

2010-01-01

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

Primate Lentiviruses Modulate NF-κB Activity by Multiple Mechanisms to Fine-Tune Viral and Cellular Gene Expression

PubMed Central

Heusinger, Elena; Kirchhoff, Frank

2017-01-01

The transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) plays a complex role during the replication of primate lentiviruses. On the one hand, NF-κB is essential for induction of efficient proviral gene expression. On the other hand, this transcription factor contributes to the innate immune response and induces expression of numerous cellular antiviral genes. Recent data suggest that primate lentiviruses cope with this challenge by boosting NF-κB activity early during the replication cycle to initiate Tat-driven viral transcription and suppressing it at later stages to minimize antiviral gene expression. Human and simian immunodeficiency viruses (HIV and SIV, respectively) initially exploit their accessory Nef protein to increase the responsiveness of infected CD4+ T cells to stimulation. Increased NF-κB activity initiates Tat expression and productive replication. These events happen quickly after infection since Nef is rapidly expressed at high levels. Later during infection, Nef proteins of HIV-2 and most SIVs exert a very different effect: by down-modulating the CD3 receptor, an essential factor for T cell receptor (TCR) signaling, they prevent stimulation of CD4+ T cells via antigen-presenting cells and hence suppress further induction of NF-κB and an effective antiviral immune response. Efficient LTR-driven viral transcription is maintained because it is largely independent of NF-κB in the presence of Tat. In contrast, human immunodeficiency virus type 1 (HIV-1) and its simian precursors have lost the CD3 down-modulation function of Nef and use the late viral protein U (Vpu) to inhibit NF-κB activity by suppressing its nuclear translocation. In this review, we discuss how HIV-1 and other primate lentiviruses might balance viral and antiviral gene expression through a tight temporal regulation of NF-κB activity throughout their replication cycle. PMID:28261165

Activation of AMPK Inhibits Cholera Toxin Stimulated Chloride Secretion in Human and Murine Intestine

PubMed Central

Hoekstra, Nadia; Collins, Danielle; Collaco, Anne; Baird, Alan W.; Winter, Desmond C.; Ameen, Nadia; Geibel, John P.; Kopic, Sascha

2013-01-01

Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness. PMID:23935921

The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

PubMed

Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

2009-06-01

Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

Occurrence of N-phenylpropenoyl-L-amino acid amides in different herbal drugs and their influence on human keratinocytes, on human liver cells and on adhesion of Helicobacter pylori to the human stomach.

PubMed

Hensel, A; Deters, A M; Müller, G; Stark, T; Wittschier, N; Hofmann, T

2007-02-01

Thirty commonly used medicinal plants were screened by a selective and specific LC-MS/MS method for the occurrence of N-phenylpropenoyl- L-amino acid amides, a new homologous class of secondary products. In 15 plants, one or more of the respective derivatives (1 to 12) were found and quantitated. Especially roots from Angelica archangelica, fruits of Cassia angustifolia, C. senna, Coriandrum sativum, leaves from Hedera helix, flowers from Lavandula spec. and from Sambucus nigra contained high amounts (1 to 11 microg/g) of mixtures of the different amides 1 to 12. For functional investigations on potential activity in cellular physiology, two amides with an aliphatic (8) and an aromatic amino acid residue (5) were used. N-(E)-Caffeic acid L-aspartic acid amide (8) and N-(E)-caffeic acid L-tryptophan amide (5) stimulated mitochondrial activity as well as the proliferation rate of human liver cells (HepG2) at 10 microg/mL significantly. When monitoring the influence of selected phase I and II metabolizing enzymes, both compounds did not influence CYP3A4 gene expression, but stimulated CYP1A2 gene expression and inhibited GST expression. Also, the proliferation of human keratinocytes (NHK) was increased up to 150% by both amides 5 and 8; this stimulation was also detectable on the level of gene expression by an up-regulation of the transcription factor STAT6. The aliphatic aspartic compound 8 showed strong antiadhesive properties on the adhesion of Helicobacter pylori to human stomach tissue.

Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells.

PubMed

Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2013-02-01

Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

USF-related transcription factor, HIV-TF1, stimulates transcription of human immunodeficiency virus-1.

PubMed

Maekawa, T; Sudo, T; Kurimoto, M; Ishii, S

1991-09-11

The transcription factor HIV-TF1, which binds to a region about 60 bp upstream from the enhancer of the human immunodeficiency virus-1 (HIV-1), was purified from human B cells. HIV-TF1 had a molecular weight of 39,000. Binding of HIV-TF1 to the HIV long terminal repeat (LTR) activated transcription from the HIV promoter in vitro. The HIV-TF1-binding site in HIV LTR was similar to the site recognized by upstream stimulatory factor (USF) in the adenovirus major late promoter. DNA-binding properties of HIV-TF1 suggested that HIV-TF1 might be identical or related to USF. Interestingly, treatment of purified HIV-TF1 by phosphatase greatly reduced its DNA-binding activity, suggesting that phosphorylation of HIV-TF1 was essential for DNA binding. The disruption of HIV-TF1-binding site induced a 60% decrease in the level of transcription from the HIV promoter in vivo. These results suggest that HIV-TF1 is involved in transcriptional regulation of HIV-1.

Cell-autonomous intracellular androgen receptor signaling drives the growth of human prostate cancer initiating cells.

PubMed

Vander Griend, Donald J; D'Antonio, Jason; Gurel, Bora; Antony, Lizamma; Demarzo, Angelo M; Isaacs, John T

2010-01-01

The lethality of prostate cancer is due to the continuous growth of cancer initiating cells (CICs) which are often stimulated by androgen receptor (AR) signaling. However, the underlying molecular mechanism(s) for such AR-mediated growth stimulation are not fully understood. Such mechanisms may involve cancer cell-dependent induction of tumor stromal cells to produce paracrine growth factors or could involve cancer cell autonomous autocrine and/or intracellular AR signaling pathways. We utilized clinical samples, animal models and a series of AR-positive human prostate cancer cell lines to evaluate AR-mediated growth stimulation of prostate CICs. The present studies document that stromal AR expression is not required for prostate cancer growth, since tumor stroma surrounding AR-positive human prostate cancer metastases (N = 127) are characteristically AR-negative. This lack of a requirement for AR expression in tumor stromal cells is also documented by the fact that human AR-positive prostate cancer cells grow equally well when xenografted in wild-type versus AR-null nude mice. AR-dependent growth stimulation was documented to involve secretion, extracellular binding, and signaling by autocrine growth factors. Orthotopic xenograft animal studies documented that the cellautonomous autocrine growth factors which stimulate prostate CIC growth are not the andromedins secreted by normal prostate stromal cells. Such cell autonomous and extracellular autocrine signaling is necessary but not sufficient for the optimal growth of prostate CICs based upon the response to anti-androgen plus/or minus preconditioned media. AR-induced growth stimulation of human prostate CICs requires AR-dependent intracellular pathways. The identification of such AR-dependent intracellular pathways offers new leads for the development of effective therapies for prostate cancer. (c) 2009 Wiley-Liss, Inc.

U-937 Toxicity Testing of Lunar Dust Stimulant (JSC-1A-vf)

NASA Technical Reports Server (NTRS)

Bales, Kristyn; Hammond, Dianne; Wallace, William; Jeevarajan, Antony

2007-01-01

With NASA planning to extend the human presence to the moon by 2020, the dangers of the lunar environment must be assessed and appropriate countermeasures must be developed. Possible toxic effects of the lunar dust are of particular importance to human health because of the dust's chemical composition, reactivity, and small size. This project focuses on the toxicity of lunar dust stimulant (JSC-1A-vf), in both its active and passive forms, using U-937 human monocyte cells. Simulant was mechanically activated from its passive form by grinding, and its ability to produce hydroxyl radicals was determined. To test for toxicity, active and passivated simulant was diluted in media and applied to the cells for various time periods. Toxicity was then estimated using flow cytometry on the Guava Personal Cell Analysis system. Preliminary results suggest that passivated stimulant is slightly toxic, with an increase in toxicity for activated stimulant. Toxicity results may be affected by cell lysing behavior and quenching of hydroxyl radical production by the cell media.

Antiapoptotic effects of Phe140Asn, a novel human granulocyte colony-stimulating factor mutant in H9c2 rat cardiomyocytes.

PubMed

Chung, Hee Kyoung; Ko, Eun Mi; Kim, Sung Woo; Byun, Sung-June; Chung, Hak-Jae; Kwon, Moosik; Lee, Hwi-Cheul; Yang, Byoung-Chul; Han, Deug-Woo; Park, Jin-Ki; Hong, Sung-Gu; Chang, Won-Kyong; Kim, Kyung-Woon

2012-12-01

Granulocyte colony-stimulating factor (G-CSF) is used for heart failure therapy and promotes myocardial regeneration by inducing mobilization of bone marrow stem cells to the injured heart after myocardial infarction; however, this treatment has one weakness in that its biological effect is transient. In our previous report, we generated 5 mutants harboring N-linked glycosylation to improve its antiapoptotic activities. Among them, one mutant (Phe140Asn) had higher cell viability than wild-type hG-CSF in rat cardiomyocytes, even after treatment with an apoptotic agent (H2O2). Cells treated with this mutant significantly upregulated the antiapoptotic proteins, and experienced reductions in caspase 3 activity and PARP cleavage. Moreover, the total number of apoptotic cells was dramatically lower in cultures treated with mutant hG-CSF. Taken together, these results suggest that the addition of an N-linked glycosylation was successful in improving the antiapoptotic activity of hG-CSF, and that this mutated product will be a feasible therapy for patients who have experienced heart failure.

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

PubMed

Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

2015-04-01

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. © 2014 John Wiley & Sons Ltd.

Exercise protects the cardiovascular system: effects beyond traditional risk factors

PubMed Central

Joyner, Michael J; Green, Daniel J

2009-01-01

In humans, exercise training and moderate to high levels of physical activity are protective against cardiovascular disease. In fact they are ∼40% more protective than predicted based on the changes in traditional risk factors (blood lipids, hypertension, diabetes etc.) that they cause. In this review, we highlight the positive effects of exercise on endothelial function and the autonomic nervous system. We also ask if these effects alone, or in combination, might explain the protective effects of exercise against cardiovascular disease that appear to be independent of traditional risk factor modification. Our goal is to use selected data from our own work and that of others to stimulate debate on the nature and cause of the ‘risk factor gap’ associated with exercise and physical activity. PMID:19736305

PI3K, ERK, p38 MAPK and integrins regulate CCR3-mediated secretion of mouse and human eosinophil-associated RNases

PubMed Central

Shamri, Revital; Young, Kristen M.; Weller, Peter F.

2013-01-01

Background Eosinophils have the capacity to secrete varied cytotoxic proteins. Among the proteins are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein and their murine ortholog EARs, which have been shown to be involved in host defense, tissue remodeling and immunity regulation. However, the signal transduction that regulates EARs secretion in response to physiological stimuli, such as chemokines, has been little studied in human and scarcely in mouse eosinophils, the foremost animal model for eosinophil-associated human diseases. Objective In this study we aimed to understand the signal transduction involved in the secretion of enzymatically active EARs following chemokine stimulation. Methods Fresh mouse and human eosinophils were stimulated with CCL11 and CCL24 and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of signaling factors or integrins was probed using specific inhibitors and blocking antibodies. Adhesion was evaluated by microscopy. Results We found that secretion of mouse EARs in response to CCL11 and CCL24 was Gαi-dependent. Both mouse and human eosinophils required the activation of PI3K, ERK and p38 MAPK. In addition, the adhesion molecules β1 and β2 integrins were found to be crucial for EAR secretion, and we suggest a mechanism in which spreading is obligatory for EAR secretion. Conclusions Collectively, these data suggest a common CCR3-mediated signaling pathway that leads to EAR secretion in both mouse and human eosinophils. These findings are applicable for eosinophil-mediated host defense and eosinophil-associated diseases. PMID:23742707

Direct anti-inflammatory effects of granulocyte colony-stimulating factor (G-CSF) on activation and functional properties of human T cell subpopulations in vitro.

PubMed

Malashchenko, Vladimir Vladimirovich; Meniailo, Maxsim Evgenievich; Shmarov, Viacheslav Anatolievich; Gazatova, Natalia Dinislamovna; Melashchenko, Olga Borisovna; Goncharov, Andrei Gennadievich; Seledtsova, Galina Victorovna; Seledtsov, Victor Ivanovich

2018-03-01

We investigated the direct effects of human granulocyte colony-stimulating factor (G-CSF) on functionality of human T-cell subsets. CD3 + T-lymphocytes were isolated from blood of healthy donors by positive magnetic separation. T cell activation with particles conjugated with antibodies (Abs) to human CD3, CD28 and CD2 molecules increased the proportion of cells expressing G-CSF receptor (G-CSFR, CD114) in all T cell subpopulations studied (CD45RA + /CD197 + naive T cells, CD45RA - /CD197 + central memory T cells, CD45RA - /CD197 - effector memory T cells and CD45RA + /CD197 - terminally differentiated effector T cells). Upon T-cell activation in vitro, G-CSF (10.0 ng/ml) significantly and specifically enhanced the proportion of CD114 + T cells in central memory CD4 + T cell compartment. A dilution series of G-CSF (range, 0.1-10.0 ng/ml) was tested, with no effect on the expression of CD25 (interleukin-2 receptor α-chain) on activated T cells. Meanwhile, G-CSF treatment enhanced the proportion of CD38 + T cells in CD4 + naïve T cell, effector memory T cell and terminally differentiated effector T cell subsets, as well as in CD4 - central memory T cells and terminally differentiated effector T cells. G-CSF did not affect IL-2 production by T cells; relatively low concentrations of G-CSF down-regulated INF-γ production, while high concentrations of this cytokine up-regulated IL-4 production in activated T cells. The data obtained suggests that G-CSF could play a significant role both in preventing the development of excessive and potentially damaging inflammatory reactivity, and in constraining the expansion of potentially cytodestructive T cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Immunomodulatory activity of polysaccharides isolated from Alchornea cordifolia

PubMed Central

Kouakou, Koffi; Schepetkin, Igor A.; Yapi, Ahoua; Kirpotina, Liliya N.; Jutila, Mark A.; Quinn, Mark T.

2013-01-01

Ethnopharmacological relevance Extracts of leaves from different species of the genus Alchornea have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts. Objective The aim of this study was to evaluate the immunomodulatory activity of polysaccharides isolated from the leaves of Alchornea cordifolia. Materials and methods Water-soluble polysaccharides from leaves of A. cordifolia were extracted and fractionated by DEAE-cellulose, Diaion HP-20, and size-exclusion chromatography. Molecular weight, sugar analysis, and other physical and chemical characterization of the fractions were performed. Immunomodulatory activity of the polysaccharide fractions was evaluated by determining their ability to induce monocyte/macrophage nitric oxide (NO) and cytokine production. Activation of mitogen activated protein kinases (MAPK) was also assessed using a phospho-MAPK array. Activation of nuclear factor κB (NF-κB) was measured using an alkaline phosphatase reporter gene assay in THP1-Blue monocytic cells. Results Six polysaccharide fractions from A. cordifolia were isolated. Fractions containing type II arabinogalactan had potent immunomodulatory activity. Particularly, the parent fraction AP-AU and its high-molecular weight sub-fraction AP-AU1 (average Mr was estimated to be 39.5 kDa) induced production of NO and cytokines [interleukin (IL)-1β, -6, -10, tumor necrosis factor (TNF)-α, and granulocyte macrophage-colony stimulating factor (GM-CSF)] in human peripheral blood mononuclear cells and human and murine monocyte/macrophages cell lines in vitro. Furthermore, treatment with AP-AU1 induced phosphorylation of Akt2, p38δ/p38γ, p70S6K1, RSK2, and mTOR, as well as stimulation of NF-κB transcriptional activity. Conclusion Our results provide a molecular basis to explain a portion of the beneficial therapeutic properties of water extracts from A. cordifolia leaves in traditional folk medicine of Africa. PMID:23291534

Immunomodulatory activity of polysaccharides isolated from Alchornea cordifolia.

PubMed

Kouakou, Koffi; Schepetkin, Igor A; Yapi, Ahoua; Kirpotina, Liliya N; Jutila, Mark A; Quinn, Mark T

2013-03-07

Extracts of leaves from different species of the genus Alchornea have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts. The aim of this study was to evaluate the immunomodulatory activity of polysaccharides isolated from the leaves of Alchornea cordifolia. Water-soluble polysaccharides from leaves of Alchornea cordifolia were extracted and fractionated by DEAE-cellulose, Diaion HP-20, and size-exclusion chromatography. Molecular weight, sugar analysis, and other physical and chemical characterization of the fractions were performed. Immunomodulatory activity of the polysaccharide fractions was evaluated by determining their ability to induce monocyte/macrophage nitric oxide (NO) and cytokine production. Activation of mitogen activated protein kinases (MAPK) was also assessed using a phospho-MAPK array. Activation of nuclear factor κB (NF-κB) was measured using an alkaline phosphatase reporter gene assay in THP1-Blue monocytic cells. Six polysaccharide fractions from Alchornea cordifolia were isolated. Fractions containing type II arabinogalactan had potent immunomodulatory activity. Particularly, the parent fraction AP-AU and its high-molecular weight sub-fraction AP-AU1 (average M(r) was estimated to be 39.5kDa) induced production of NO and cytokines [interleukin (IL)-1β, -6, -10, tumor necrosis factor (TNF)-α, and granulocyte-macrophage-colony stimulating factor (GM-CSF)] in human peripheral blood mononuclear cells and human and murine monocyte/macrophages cell lines in vitro. Furthermore, treatment with AP-AU1 induced phosphorylation of Akt2, p38δ/p38γ, p70S6K1, RSK2, and mTOR, as well as stimulation of NF-κB transcriptional activity. Our results provide a molecular basis to explain a portion of the beneficial therapeutic properties of water extracts from Alchornea cordifolia leaves in traditional folk medicine of Africa. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Sema4D, the ligand for Plexin B1, suppresses c-Met activation and migration and promotes melanocyte survival and growth

PubMed Central

Soong, Joanne; Chen, Yulin; Shustef, Elina; Scott, Glynis

2011-01-01

Semaphorins are secreted and membrane bound proteins involved in neural pathfinding, organogenesis, and tumor progression, through Plexin and neuropilins receptors. We recently reported that Plexin B1, the Semaphorin 4D receptor, is a tumor suppressor protein for melanoma, in part, through inhibition of the oncogenic c-Met tyrosine kinase receptor. In this report we show that Sema4D is a protective paracrine factor for normal human melanocyte survival in response to ultraviolet irradiation, that it stimulates proliferation, and regulates the activity of the c-Met receptor. c-Met receptor signaling stimulates melanocyte migration, in part through down-regulation of the cell adhesion molecule E-cadherin. Sema4D suppressed activation of c-Met in response to its ligand hepatocyte growth factor (HGF), and partially blocked the suppressive effects of HGF on E-cadherin expression in melanocytes and HGF-dependent migration. These data demonstrate a role for Plexin B1 in maintenance of melanocyte survival and proliferation in the skin, and suggest that Semaphorin 4D and Plexin B1 act cooperatively with HGF and c-Met to regulate c-Met dependent effects in human melanocytes. Because our data show that Plexin B1 is profoundly down-regulated by UVB in melanocytes, loss of Plexin B1 may accentuate HGF dependent effects on melanocytes, including melanocyte migration. PMID:22189792

Enhanced heterologous expression of biologically active human granulocyte colony stimulating factor in transgenic tobacco BY-2 cells by localization to endoplasmic reticulum.

PubMed

Nair, Nisha R; Chidambareswaren, M; Manjula, S

2014-09-01

Tobacco Bright Yellow-2 (BY-2) cells, one of the best characterized cell lines is an attractive expression system for heterologous protein expression. However, the expression of foreign proteins is currently hampered by their low yield, which is partially the result of proteolytic degradation. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine. Recombinant hG-CSF is successfully being used for the treatment of chemotherapy-induced neutropenia in cancer patients. Here, we describe a simple strategy for producing biologically active hG-CSF in tobacco BY-2 cells, localized in the apoplast of BY-2 cells, as well as targeted to the endoplasmic reticulum (ER). ER targeting significantly enhanced recombinant production which scaled to 17.89 mg/l from 4.19 mg/l when expressed in the apoplasts. Southern blotting confirmed the stable integration of hG-CSF in the BY-2 nuclear genome, and the expression of hG-CSF was analysed by Western blotting. Total soluble protein containing hG-CSF isolated from positive calli showed proliferative potential when tested on HL-60 cell lines by MTT assay. We also report the potential of a Fluorescence-activated cell sorting approach for an efficient sorting of the hG-CSF-expressing cell lines, which will enable the generation of homogenous high-producing cell lines.

Sema4D, the ligand for Plexin B1, suppresses c-Met activation and migration and promotes melanocyte survival and growth.

PubMed

Soong, Joanne; Chen, Yulin; Shustef, Elina M; Scott, Glynis A

2012-04-01

Semaphorins are secreted and membrane-bound proteins involved in neural pathfinding, organogenesis, and tumor progression, through Plexin and neuropilin receptors. We recently reported that Plexin B1, the Semaphorin 4D (Sema4D) receptor, is a tumor-suppressor protein for melanoma, which functions, in part, through inhibition of the oncogenic c-Met tyrosine kinase receptor. In this report, we show that Sema4D is a protective paracrine factor for normal human melanocyte survival in response to UV irradiation, and that it stimulates proliferation and regulates the activity of the c-Met receptor. c-Met receptor signaling stimulates melanocyte migration, partly through downregulation of the cell adhesion molecule E-cadherin. Sema4D suppressed activation of c-Met in response to its ligand, hepatocyte growth factor (HGF), and partially blocked the suppressive effects of HGF on E-cadherin expression in melanocytes and HGF-dependent migration. These data demonstrate a role for Plexin B1 in maintenance of melanocyte survival and proliferation in the skin, and suggest that Sema4D and Plexin B1 act cooperatively with HGF and c-Met to regulate c-Met-dependent effects in human melanocytes. Because our data show that Plexin B1 is profoundly downregulated by UVB in melanocytes, loss of Plexin B1 may accentuate HGF-dependent effects on melanocytes, including melanocyte migration.

Immunomodulatory Effects of the Mycosporine-Like Amino Acids Shinorine and Porphyra-334

PubMed Central

Becker, Kathrin; Hartmann, Anja; Ganzera, Markus; Fuchs, Dietmar; Gostner, Johanna M.

2016-01-01

Mycosporine-like amino acids (MAAs) are secondary metabolites, produced by a large variety of microorganisms including algae, cyanobacteria, lichen and fungi. MAAs act as UV-absorbers and photo-protectants. MAAs are suggested to exert pharmaceutical relevant bioactivities in the human system. We particularly focused on their effect on defence and regulatory pathways that are active in inflamed environments. The MAAs shinorine and porphyra-334 were isolated and purified from the red algae Porphyra sp. using chromatographic methods. The effect of MAAs on central signaling cascades, such as transcription factor nuclear factor kappa b (NF-κB) activation, as well as tryptophan metabolism, was investigated in human myelomonocytic THP-1 and THP-1-Blue cells. Cells were exposed to the MAAs in the presence or absence of lipopolysaccharide (LPS). NF-κB activity and the activity of tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO-1) were used as readout. Compounds were tested in the concentration range from 12.5 to 200 µg/mL. Both MAAs were able to induce NF-κB activity in unstimulated THP-1-Blue cells, whereby the increase was dose-dependent and more pronounced with shinorine treatment. While shinorine also slightly superinduced NF-κB in LPS-stimulated cells, porphyra-334 reduced NF-κB activity in this inflammatory background. Modulation of tryptophan metabolism was moderate, suppressive in stimulated cells with the lower treatment concentration of both MAAs and with the unstimulated cells upon porphyra-334 treatment. Inflammatory pathways are affected by MAAs, but despite the structural similarity, diverse effects were observed. PMID:27338421

HILDA/LIF urinary excretion during acute kidney rejection.

PubMed

Taupin, J L; Morel, D; Moreau, J F; Gualde, N; Potaux, L; Bezian, J H

1992-03-01

Recently, a new lymphokine called HILDA (human interleukin for DA cells) has been described and cloned. This cytokine, initially described to be produced by alloreactive T lymphocyte clones grown from a rejected human kidney allograft, is identical to other factors termed D-factor, differentiation-inducing factor, differentiation inhibitory activity, hepatocyte-stimulating factor III, and leukemia inhibitory factor. HILDA/LIF induces various effects on neural, hemopoietic, embryonic cells as well as on bone remodeling and acute phase protein synthesis in hepatocyte. In this study we demonstrate the presence of HILDA/LIF in the urine but not in the serum of kidney graft recipients during acute rejection episodes, whereas this lymphokine was detectable neither in the serum nor in the urine of kidney transplanted patients with stable renal function. These data reinforce the notion of a possible role for this lymphokine in the inflammatory and/or the immune response.

Noradrenaline inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin 6 production in human whole blood.

PubMed Central

van der Poll, T; Jansen, J; Endert, E; Sauerwein, H P; van Deventer, S J

1994-01-01

Sepsis and lipopolysaccharide (LPS) trigger the systemic release of both cytokines and catecholamines. Cytokines are known to be capable of eliciting a stress hormone response in vivo. The present study sought insight into the effect of noradrenaline on LPS-induced release of tumor necrosis factor alpha (TNF) and interleukin 6 (IL-6) in human whole blood. Whole blood was incubated with LPS for 4 h at 37 degrees C in the presence and absence of noradrenaline and/or specific alpha and beta antagonists and agonists. Noradrenaline caused a dose-dependent inhibition of LPS-induced TNF and IL-6 production. This effect could be completely prevented by addition of the specific beta 1, antagonist metoprolol, while it was not affected by the alpha antagonist phentolamine. Specific beta-adrenergic stimulation by isoprenaline mimicked the inhibiting effect of noradrenaline on LPS-evoked cytokine production, whereas alpha-adrenergic stimulation by phenylephrine had no effect. Fluorescence-activated cell sorter analysis demonstrated that beta-adrenergic stimulation had no effect on LPS binding to and internalization into mononuclear cells or on the expression of CD14, the major receptor for LPS on mononuclear cells. In acute sepsis, enhanced release of noradrenaline may be part of a negative feedback mechanism meant to inhibit ongoing TNF and IL-6 production. PMID:8168970

EFFECTS OF MUSIC ON WORK PERFORMANCE.

DTIC Science & Technology

MUSIC , *HUMAN FACTORS ENGINEERING), (*ATTENTION, PERFORMANCE(HUMAN)), REACTION(PSYCHOLOGY), REFLEXES, ATTITUDES(PSYCHOLOGY), QUESTIONNAIRES, STIMULATION(PHYSIOLOGY), EXPERIMENTAL DESIGN, CORRELATION TECHNIQUES

Human Natural Killer Cells Exhibit Direct Activity Against Aspergillus fumigatus Hyphae, But Not Against Resting Conidia

PubMed Central

Schmidt, Stanislaw; Tramsen, Lars; Hanisch, Mitra; Latgé, Jean-Paul; Huenecke, Sabine; Koehl, Ulrike

2011-01-01

Because natural killer (NK) cells kill tumor cells and combat infections, there is growing interest in adoptively transferring NK cells to hematopoietic stem cell recipients. Unfortunately, in humans, the activity of NK cells against Aspergillus species, the major cause of invasive fungal infection in stem cell recipients, are poorly characterized. Our results show that unstimulated and interleukin-2 prestimulated human NK cells kill Aspergillus fumigatus hyphae but do not affect resting conidia. Killing is also induced by the supernatant of prestimulated NK cells and human perforin. The high levels of interferon-γ and granulocyte macrophage colony-stimulating factor produced by prestimulated NK cells are significantly reduced by Aspergillus, indicating an immunosuppressive effect of the fungus. Whereas Aspergillus hyphae activate NK cells, resting, and germinating, conidia and conidia of ΔrodA mutants lacking the hydrophobic surface layer do not. Our results suggest that adoptively transferred human NK cells may be a potential antifungal tool in the transplantation context. PMID:21208932

Sex-dependent differences in voluntary physical activity.

PubMed

Rosenfeld, Cheryl S

2017-01-02

Numbers of overweight and obese individuals are increasing in the United States and globally, and, correspondingly, the associated health care costs are rising dramatically. More than one-third of children are currently considered obese with a predisposition to type 2 diabetes, and it is likely that their metabolic conditions will worsen with age. Physical inactivity has also risen to be the leading cause of many chronic, noncommunicable diseases (NCD). Children are more physically inactive now than they were in past decades, which may be due to intrinsic and extrinsic factors. In rodents, the amount of time engaged in spontaneous activity within the home cage is a strong predictor of later adiposity and weight gain. Thus, it is important to understand primary motivators stimulating physical activity (PA). There are normal sex differences in PA levels in rodents and humans. The perinatal environment can induce sex-dependent differences in PA disturbances. This Review considers the current evidence for sex differences in PA in rodents and humans. The rodent studies showing that early exposure to environmental chemicals can shape later adult PA responses are discussed. Next, whether there are different motivators stimulating exercise in male vs. female humans are examined. Finally, the brain regions, genes, and pathways that modulate PA in rodents, and possibly by translation in humans, are described. A better understanding of why each sex remains physically active through the life span could open new avenues for preventing and treating obesity in children and adults. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

PubMed

Zhang, Feng; Shu, Jin-Ling; Li, Ying; Wu, Yu-Jing; Zhang, Xian-Zheng; Han, Le; Tang, Xiao-Yu; Wang, Chen; Wang, Qing-Tong; Chen, Jing-Yu; Chang, Yan; Wu, Hua-Xun; Zhang, Ling-Ling; Wei, Wei

2017-01-01

Paeoniflorin-6'- O -benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 + B cells, CD19 + CD20 + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

Aldosterone stimulates vacuolar H+-ATPase activity in renal acid-secretory intercalated cells mainly via a protein kinase C-dependent pathway

PubMed Central

Winter, Christian; Kampik, Nicole B.; Vedovelli, Luca; Rothenberger, Florina; Păunescu, Teodor G.; Stehberger, Paul A.; Brown, Dennis; John, Hubert

2011-01-01

Urinary acidification in the collecting duct is mediated by the activity of H+-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H+-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H+-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific Gαq inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca2+ with BAPTA, and blockade of protein kinase C prevented the stimulation of H+-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H+-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H+-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H+-ATPase activity. Thus, the nongenomic modulation of H+-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a Gαq protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H+-ATPase activity and contribute to final urinary acidification. PMID:21832245

Neurotrophin-3 accelerates wound healing in diabetic mice by promoting a paracrine response in mesenchymal stem cells.

PubMed

Shen, Lei; Zeng, Wen; Wu, Yang-Xiao; Hou, Chun-Li; Chen, Wen; Yang, Ming-Can; Li, Li; Zhang, Ya-Fang; Zhu, Chu-Hong

2013-01-01

Angiogenesis is a major obstacle for wound healing in patients with diabetic foot wounds. Mesenchymal stem cells (MSCs) have an important function in wound repair, and neurotrophin-3 (NT-3) can promote nerve regeneration and angiogenesis. We investigated the effect of NT-3 on accelerating wound healing in the diabetic foot by improving human bone marrow MSC (hMSC) activation. In vitro, NT-3 significantly promoted VEGF, NGF, and BDNF secretion in hMSCs. NT-3 improved activation of the hMSC conditioned medium, promoted human umbilical vein endothelial cell (HUVEC) proliferation and migration, and significantly improved the closure rate of HUVEC scratches. In addition, we produced nanofiber mesh biological tissue materials through the electrospinning technique using polylactic acid, mixed silk, and collagen. The hMSCs stimulated by NT-3 were implanted into the material. Compared with the control group, the NT-3-stimulated hMSCs in the biological tissue material significantly promoted angiogenesis in the feet of diabetic C57BL/6J mice and accelerated diabetic foot wound healing. These results suggest that NT-3 significantly promotes hMSC secretion of VEGF, NGF, and other vasoactive factors and that it accelerates wound healing by inducing angiogenesis through improved activation of vascular endothelial cells. The hMSCs stimulated by NT-3 can produce materials that accelerate wound healing in the diabetic foot and other ischemic ulcers.

Antagonist interaction with the human 5-HT7 receptor mediates the rapid and potent inhibition of non-G-protein-stimulated adenylate cyclase activity: a novel GPCR effect

PubMed Central

Klein, MT; Teitler, M

2011-01-01

BACKGROUND AND PURPOSE The human 5-hydroxytryptamine7 (h5-HT7) receptor is Gs-coupled and stimulates the production of the intracellular signalling molecule cAMP. Previously, we reported a novel property of the h5-HT7 receptor: pseudo-irreversible antagonists irreversibly inhibit forskolin-stimulated (non-receptor-mediated) cAMP production. Herein, we sought to determine if competitive antagonists also affect forskolin-stimulated activity and if this effect is common among other Gs-coupled receptors. EXPERIMENTAL APPROACH Recombinant cell lines expressing h5-HT7 receptors or other receptors of interest were briefly exposed to antagonists; cAMP production was then stimulated by forskolin and quantified by an immunocompetitive assay. KEY RESULTS In human embryonic kidney 293 cells stably expressing h5-HT7 receptors, all competitive antagonists inhibited nearly 100% of forskolin-stimulated cAMP production. This effect was insensitive to pertussis toxin, that is, not Gi/o-mediated. Potency to inhibit forskolin-stimulated activity strongly correlated with h5-HT7 binding affinity (r2= 0.91), indicating that the antagonists acted through h5-HT7 receptors to inhibit forskolin. Potency and maximal effects of clozapine, a prototypical competitive h5-HT7 antagonist, were unaffected by varying forskolin concentration. Antagonist interaction with h5-HT6, human β1, β2, and β3 adrenoceptors did not inhibit forskolin's activity. CONCLUSIONS AND IMPLICATIONS The inhibition of adenylate cyclase, as measured by forskolin's activity, is an underlying property of antagonist interaction with h5-HT7 receptors; however, this is not a common property of other Gs-coupled receptors. This phenomenon may be involved in the roles played by h5-HT7 receptors in human physiology. Development of h5-HT7 antagonists that do not elicit this effect would aid in the elucidation of its mechanisms and shed light on its possible physiological relevance. PMID:21198551

PKC-dependent stimulation of the human MCT1 promoter involves transcription factor AP2.

PubMed

Saksena, Seema; Dwivedi, Alka; Gill, Ravinder K; Singla, Amika; Alrefai, Waddah A; Malakooti, Jaleh; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

2009-02-01

Monocarboxylate transporter (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFA) such as butyrate in the human colon. Previous studies from our laboratory have demonstrated that phorbol ester, PMA (1 microM, 24 h), upregulates butyrate transport and MCT1 protein expression in human intestinal Caco-2 cells. However, the molecular mechanisms involved in the transcriptional regulation of MCT1 gene expression by PMA in the intestine are not known. In the present study, we showed that PMA (0.1 microM, 24 h) increased the MCT1 promoter activity (-871/+91) by approximately fourfold. A corresponding increase in MCT1 mRNA abundance in response to PMA was also observed. PMA-induced stimulation of MCT1 promoter activity was observed as early as 1 h and persisted until 24 h, suggesting that the effects of PMA are attributable to initial PKC activation. Kinase inhibitor and phosphorylation studies indicated that these effects may be mediated through activation of the atypical PKC-zeta isoform. 5'-deletion studies demonstrated that the MCT1 core promoter region (-229/+91) is the PMA-responsive region. Site-directed mutagenesis studies showed the predominant involvement of potential activator protein 2 (AP2) binding site in the activation of MCT1 promoter activity by PMA. In addition, overexpression of AP2 in Caco-2 cells significantly increased MCT1 promoter activity in a dose-dependent manner. These findings showing the regulation of MCT1 promoter by PKC and AP2 are of significant importance for an understanding of the molecular regulation of SCFA absorption in the human intestine.

Pregnancy Augments VEGF-Stimulated In Vitro Angiogenesis and Vasodilator (NO and H2S) Production in Human Uterine Artery Endothelial Cells.

PubMed

Zhang, Hong-Hai; Chen, Jennifer C; Sheibani, Lili; Lechuga, Thomas J; Chen, Dong-Bao

2017-07-01

Augmented uterine artery (UA) production of vasodilators, including nitric oxide (NO) and hydrogen sulfide (H2S), has been implicated in pregnancy-associated and agonist-stimulated rise in uterine blood flow that is rate-limiting to pregnancy health. Developing a human UA endothelial cell (hUAEC) culture model from main UAs of nonpregnant (NP) and pregnant (P) women for testing a hypothesis that pregnancy augments endothelial NO and H2S production and endothelial reactivity to vascular endothelial growth factor (VEGF). Main UAs from NP and P women were used for developing hUAEC culture models. Comparisons were made between NP- and P-hUAECs in in vitro angiogenesis, activation of cell signaling, expression of endothelial NO synthase (eNOS) and H2S-producing enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase, and NO/H2S production upon VEGF stimulation. NP- and P-hUAECs displayed a typical cobblestone-like shape in culture and acetylated low-density lipoprotein uptake, stained positively for endothelial and negatively for smooth muscle markers, maintained key signaling proteins during passage, and had statistically significant greater eNOS and CBS proteins in P- vs NP-hUAECs. Treatment with VEGF stimulated in vitro angiogenesis and eNOS protein and NO production only in P-hUEACs and more robust cell signaling in P- vs NP-hUAECs. VEGF stimulated CBS protein expression, accounting for VEGF-stimulated H2S production in hUAECs. Comparisons between NP- and P-hUAECs reveal that pregnancy augments VEGF-stimulated in vitro angiogenesis and NO/H2S production in hUAECs, showing that the newly established hUAEC model provides a critical in vitro tool for understanding human uterine hemodynamics. Copyright © 2017 Endocrine Society

A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.

PubMed

Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J V; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

2013-07-08

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Comparative effects of sub-stimulating concentrations of non-human versus human Luteinizing Hormones (LH) or chorionic gonadotropins (CG) on adenylate cyclase activation by forskolin in MLTC cells.

PubMed

Nguyen, Thi-Mong Diep; Filliatreau, Laura; Klett, Danièle; Combarnous, Yves

2018-05-15

We have compared various Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) preparations from non-human and human species in their ability to synergize with 10 µM forskolin (FSK) for cyclic AMP intracellular accumulation, in MLTC cells. LH from rat pituitary as well as various isoforms of pituitary ovine, bovine, porcine, equine and human LHs and equine and human CG were studied. In addition, recombinant human LH and CG were also compared with the natural human and non-human hormones. Sub-stimulating concentrations of all LHs and CGs (2-100 pM) were found to stimulate cyclic AMP accumulation in MLTC cells in the presence of an also non-stimulating FSK concentration (10 µM). Like rat LH, the most homologous available hormone for mouse MLTC cells, all non-human LHs and CG exhibit a strong potentiating effect on FSK response. The human, natural and recombinant hLH and hCG also do so but in addition, they were found to elicit a permissive effect on FSK stimulation. Indeed, when incubated alone with MLTC cells at non-stimulating concentrations (2-70 pM) hLH and hCG permit, after being removed, a dose-dependent cyclic AMP accumulation with 10 µM FSK. Our data show a clearcut difference between human LH and CG compared to their non-human counterparts on MLTC cells adenylate cyclase activity control. This points out the risk of using hCG as a reference ligand for LHR in studies using non-human cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets.

PubMed

Moore, S F; Hunter, R W; Hers, I

2014-05-01

Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)-stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y(12). PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y(12) antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y(12), in a manner largely independent of the canonical PI3 kinase/Akt pathway. © 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Protein kinase C and P2Y12 take center stage in thrombin-mediated activation of mammalian target of rapamycin complex 1 in human platelets

PubMed Central

Moore, S F; Hunter, R W; Hers, I

2014-01-01

Background Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets. Methods Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K). Results Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited. Conclusion These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner largely independent of the canonical PI3 kinase/Akt pathway. PMID:24612393

The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yue; Hu, Yali; Zhao, Jing

2011-01-14

Research highlights: {yields} Decidually produced PRL plays a key role during pregnancy. {yields} Overexpression of Nur77 increased PRL mRNA expression and enhanced decidual PRL promoter activity. {yields} Knockdown of Nur77 decreased decidual PRL secretion induced by 8-Br-cAMP and MPA. {yields} Nur77 is a novel transcription factor that plays an active role in decidual prolactin expression. -- Abstract: Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence thatmore » the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.« less

wALADin benzimidazoles differentially modulate the function of porphobilinogen synthase orthologs.

PubMed

Lentz, Christian S; Halls, Victoria S; Hannam, Jeffrey S; Strassel, Silke; Lawrence, Sarah H; Jaffe, Eileen K; Famulok, Michael; Hoerauf, Achim; Pfarr, Kenneth M

2014-03-27

The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg(2+), or K(+) stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design of tailored PBGS modulators for potential application as antimicrobial agents, herbicides, or drugs for porphyric disorders.

wALADin Benzimidazoles Differentially Modulate the Function of Porphobilinogen Synthase Orthologs

PubMed Central

2015-01-01

The heme biosynthesis enzyme porphobilinogen synthase (PBGS) is a potential drug target in several human pathogens. wALADin1 benzimidazoles have emerged as species-selective PBGS inhibitors against Wolbachia endobacteria of filarial worms. In the present study, we have systematically tested wALADins against PBGS orthologs from bacteria, protozoa, metazoa, and plants to elucidate the inhibitory spectrum. However, the effect of wALADin1 on different PBGS orthologs was not limited to inhibition: several orthologs were stimulated by wALADin1; others remained unaffected. We demonstrate that wALADins allosterically modulate the PBGS homooligomeric equilibrium with inhibition mediated by favoring low-activity oligomers, while 5-aminolevulinic acid, Mg2+, or K+ stabilized high-activity oligomers. Pseudomonas aeruginosa PBGS could be inhibited or stimulated by wALADin1 depending on these factors and pH. We have defined the wALADin chemotypes responsible for either inhibition or stimulation, facilitating the design of tailored PBGS modulators for potential application as antimicrobial agents, herbicides, or drugs for porphyric disorders. PMID:24568185

Helicobacter pylori neutrophil activating protein as target for new drugs against H. pylori inflammation.

PubMed

Choli-Papadopoulou, Theodora; Kottakis, Filippos; Papadopoulos, Georgios; Pendas, Stefanos

2011-06-07

Helicobacter pylori (H. pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. Within this work we present the implication of C-terminal region of H. pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evidence that the C-terminal region of H. pylori activating protein is indispensable for neutrophil adhesion to endothelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins derived from chios mastic gum, the natural resin of the plant Pistacia lentiscus var. Chia inhibit neutrophil activation in vitro.

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

NASA Astrophysics Data System (ADS)

Wu, Riqin; Zhang, Peijun; Li, Jun; Xu, Yongli

2005-03-01

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes ( P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

PubMed

Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

2016-09-15

We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. Copyright © 2016. Published by Elsevier B.V.

The human respiratory gate

NASA Technical Reports Server (NTRS)

Eckberg, Dwain L.

2003-01-01

Respiratory activity phasically alters membrane potentials of preganglionic vagal and sympathetic motoneurones and continuously modulates their responsiveness to stimulatory inputs. The most obvious manifestation of this 'respiratory gating' is respiratory sinus arrhythmia, the rhythmic fluctuations of electrocardiographic R-R intervals observed in healthy resting humans. Phasic autonomic motoneurone firing, reflecting the throughput of the system, depends importantly on the intensity of stimulatory inputs, such that when levels of stimulation are low (as with high arterial pressure and sympathetic activity, or low arterial pressure and vagal activity), respiratory fluctuations of sympathetic or vagal firing are also low. The respiratory gate has a finite capacity, and high levels of stimulation override the ability of respiration to gate autonomic responsiveness. Autonomic throughput also depends importantly on other factors, including especially, the frequency of breathing, the rate at which the gate opens and closes. Respiratory sinus arrhythmia is small at rapid, and large at slow breathing rates. The strong correlation between systolic pressure and R-R intervals at respiratory frequencies reflects the influence of respiration on these two measures, rather than arterial baroreflex physiology. A wide range of evidence suggests that respiratory activity gates the timing of autonomic motoneurone firing, but does not influence its tonic level. I propose that the most enduring significance of respiratory gating is its use as a precisely controlled experimental tool to tease out and better understand otherwise inaccessible human autonomic neurophysiological mechanisms.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

PubMed Central

Kim, Nan-Sun; Mbongue, Jacques C.; Nicholas, Dequina A.; Esebanmen, Grace E.; Unternaehrer, Juli J.; Firek, Anthony F.; Langridge, William H. R.

2016-01-01

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity. PMID:26881431

Prostaglandin E₂ regulates cellular migration via induction of vascular endothelial growth factor receptor-1 in HCA-7 human colon cancer cells.

PubMed

Fujino, Hiromichi; Toyomura, Kaori; Chen, Xiao-bo; Regan, John W; Murayama, Toshihiko

2011-02-01

An important event in the development of tumors is angiogenesis, or the formation of new blood vessels. Angiogenesis is also known to be involved in tumor cell metastasis and is dependent upon the activity of the vascular endothelial growth factor (VEGF) signaling pathway. Studies of mice in which the EP3 prostanoid receptors have been genetically deleted have shown a role for these receptors in cancer growth and angiogenesis. In the present study, human colon cancer HCA-7 cells were used as a model system to understand the potential role of EP3 receptors in tumor cell migration. We now show that stimulation of HCA-7 cells with PGE₂ enhanced the up-regulation of VEGF receptor-1 (VEGFR-1) expression by a mechanism involving EP3 receptor-mediated activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Moreover, the PGE₂ stimulated increase in VEGFR-1 expression was accompanied by an increase in the cellular migration of HCA-7 cells. Given the known involvement of VEGFR-1 in cellular migration, our results suggest that EP3 receptors may contribute to tumor cell metastasis by increasing cellular migration through the up-regulation of VEGFR-1 signaling. Copyright © 2010 Elsevier Inc. All rights reserved.

Eccrine Sweat Contains IL-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal

PubMed Central

Dai, Xiuju; Okazaki, Hidenori; Hanakawa, Yasushi; Murakami, Masamoto; Tohyama, Mikiko; Shirakata, Yuji; Sayama, Koji

2013-01-01

Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal. PMID:23874436

Staurosporine potentiates platelet activating factor stimulated phospholipase C activity in rabbit platelets but does not block desensitization by platelet activating factor.

PubMed

Morrison, W J; Dhar, A; Shukla, S D

1989-01-01

The possible involvement of protein kinase C activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets. PAF (100 nM for 5 seconds) stimulated incorporation of 32P into proteins and caused [3H]InsP3 levels to increase about 260% of control. These responses were compared after platelets were pretreated with either PAF, phorbol 12-myristate 13-acetate (PMA) or staurosporine and also after pretreatments with staurosporine followed by PAF or PMA. Pretreating platelets with staurosporine potentiated PAF-stimulated [3H]InsP3 levels by 54% and blocked protein phosphorylation. Pretreatments with PAF and PMA caused PAF-stimulated [3H]InsP3 levels to decrease to 115 and 136%, respectively. Staurosporine pretreatment blocked the decrease caused by the PMA pretreatment but not that by PAF. This study demonstrates that PAF-stimulated PLC activity is negatively affected by protein kinase C (PKC) activation and that inhibition of PKC activity did not prevent desensitization of PLC by PAF.

Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κB in airway smooth muscle cells

PubMed Central

Matsukura, S.; Odaka, M.; Kurokawa, M.; Kuga, H.; Homma, T.; Takeuchi, H.; Notomi, K.; Kokubu, F.; Kawaguchi, M.; Schleimer, R. P.; Johnson, M. W.; Adachi, M.

2013-01-01

Summary Background Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-β) may be involved in the process of airway remodelling. Objective We analysed the effects of TGF-β on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. Methods HASM cells were cultured and treated with TGF-β and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. Results IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-β alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-β. Activation by IL-4 or IL-4 plus TGF-β was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-β was inhibited by mutation of the binding site for nuclear factor-κB (NF-κB) in the promoter. Pretreatment with an inhibitor of NF-κB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-β, indicating the importance of NF-κB in the cooperative activation of CCL11 transcription by TGF-β and IL-4. Conclusion These results indicate that Th2 cytokines and TGF-β may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-κB may play pivotal roles in this process. PMID:20214667

Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κβ in airway smooth muscle cells.

PubMed

Matsukura, S; Odaka, M; Kurokawa, M; Kuga, H; Homma, T; Takeuchi, H; Notomi, K; Kokubu, F; Kawaguchi, M; Schleimer, R P; Johnson, M W; Adachi, M

2010-05-01

Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling. We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4. These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells.

PubMed

Pacini, Stefania; Punzi, Tiziana; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco

2012-01-01

Searching for additional therapeutic tools to fight breast cancer, we investigated the effects of vitamin D-binding protein-derived macrophage activating factor (DBP-MAF, also known as GcMAF) on a human breast cancer cell line (MCF-7). The effects of DBP-MAF on proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting, and chorioallantoic membrane (CAM) assay. DBP-MAF inhibited human breast cancer cell proliferation and cancer cell-stimulated angiogenesis. MCF-7 cells treated with DBP-MAF predominantly grew in monolayer and appeared to be well adherent to each other and to the well surface. Exposure to DBP-MAF significantly reduced vimentin expression, indicating a reversal of the epithelial/mesenchymal transition, a hallmark of human breast cancer progression. These results are consistent with the hypothesis that the known anticancer efficacy of DBP-MAF can be ascribed to different biological properties of the molecule that include inhibition of tumour-induced angiogenesis and direct inhibition of cancer cell proliferation, migration and metastatic potential.

A simple and quantitative liquid culture system to measure megakaryocyte growth using highly purified CFU-MK.

PubMed

Miyazaki, H; Horie, K; Shimada, Y; Kokubo, A; Maeda, E; Inoue, H; Kato, T

1995-10-01

A new and quantitative liquid culture system has been developed to measure the production of megakaryocytes from megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]). The system uses as a target population a glycoprotein (Gp) IIb/IIIa+ subpopulation of rat bone marrow cells previously demonstrated to be highly enriched for CFU-MK. GpIIb/IIIa+ cells were cultured at 5 x 10(4) cells/mL (10(4) cells/well) with test samples in 96-well tissue culture plates for 4 days at 37 degrees C. During the final 3 hours of incubation, the cells were pulsed with [14C]5-hydroxytryptamine creatinine sulfate (14C-serotonin). After incubation, the plates were washed and the cell pellets were lysed with Triton-X 100. The cell lysate was infiltrated into a commercially available solid scintillator and dried, and radioactivity was measured. In this assay system, rat interleukin-3 (IL-3) was found to be the most potent among known cytokines tested. Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), human erythropoietin (Epo), human IL-6, and murine stem cell factor (SCF) each alone stimulated megakaryocyte growth but were much less active than rat IL-3. Plasma of rats rendered thrombocytopenic by injection of monoclonal antirat platelet GpIIb/IIIa antibody exhibited significant activity, and the active protein fractions partially purified from the plasma showed much higher activity, but normal rat plasma had no effect. This liquid culture system allows the measurement of a large number of test samples--including a wide variety of cytokines and unknown growth factors, alone or in combinations--and provides a simple method for evaluating the early proliferative events involving CFU-MK in the megakaryocyte differentiation pathway.

Anti-fibrotic efficacy of nintedanib in pulmonary fibrosis via the inhibition of fibrocyte activity.

PubMed

Sato, Seidai; Shinohara, Shintaro; Hayashi, Shinya; Morizumi, Shun; Abe, Shuichi; Okazaki, Hiroyasu; Chen, Yanjuan; Goto, Hisatsugu; Aono, Yoshinori; Ogawa, Hirohisa; Koyama, Kazuya; Nishimura, Haruka; Kawano, Hiroshi; Toyoda, Yuko; Uehara, Hisanori; Nishioka, Yasuhiko

2017-09-15

Nintedanib, a tyrosine kinase inhibitor that is specific for platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR), and vascular endothelial growth factor receptors (VEGFR), has recently been approved for idiopathic pulmonary fibrosis. Fibrocytes are bone marrow-derived progenitor cells that produce growth factors and contribute to fibrogenesis in the lungs. However, the effects of nintedanib on the functions of fibrocytes remain unclear. Human monocytes were isolated from the peripheral blood of healthy volunteers. The expression of growth factors and their receptors in fibrocytes was analyzed using ELISA and Western blotting. The effects of nintedanib on the ability of fibrocytes to stimulate lung fibroblasts were examined in terms of their proliferation. The direct effects of nintedanib on the differentiation and migration of fibrocytes were also assessed. We investigated whether nintedanib affected the accumulation of fibrocytes in mouse lungs treated with bleomycin. Human fibrocytes produced PDGF, FGF2, and VEGF-A. Nintedanib and specific inhibitors for each growth factor receptor significantly inhibited the proliferation of lung fibroblasts stimulated by the supernatant of fibrocytes. Nintedanib inhibited the migration and differentiation of fibrocytes induced by growth factors in vitro. The number of fibrocytes in the bleomycin-induced lung fibrosis model was reduced by the administration of nintedanib, and this was associated with anti-fibrotic effects. These results support the role of fibrocytes as producers of and responders to growth factors, and suggest that the anti-fibrotic effects of nintedanib are at least partly mediated by suppression of fibrocyte function.

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

PubMed Central

Price, B D; Morris, J D; Hall, A

1989-01-01

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase. PMID:2690829

A functional electrical stimulation system for human walking inspired by reflexive control principles.

PubMed

Meng, Lin; Porr, Bernd; Macleod, Catherine A; Gollee, Henrik

2017-04-01

This study presents an innovative multichannel functional electrical stimulation gait-assist system which employs a well-established purely reflexive control algorithm, previously tested in a series of bipedal walking robots. In these robots, ground contact information was used to activate motors in the legs, generating a gait cycle similar to that of humans. Rather than developing a sophisticated closed-loop functional electrical stimulation control strategy for stepping, we have instead utilised our simple reflexive model where muscle activation is induced through transfer functions which translate sensory signals, predominantly ground contact information, into motor actions. The functionality of the functional electrical stimulation system was tested by analysis of the gait function of seven healthy volunteers during functional electrical stimulation-assisted treadmill walking compared to unassisted walking. The results demonstrated that the system was successful in synchronising muscle activation throughout the gait cycle and was able to promote functional hip and ankle movements. Overall, the study demonstrates the potential of human-inspired robotic systems in the design of assistive devices for bipedal walking.

Human Factors Topics in Flight Simulation: An Annotated Bibliography

DTIC Science & Technology

1976-01-01

and technical information; - Continuously stimulating advances in the aerospace sciences relevant to strengthening the common defence posture...Topics treated under cost effectiveness are effectiveness as a function of training procedures, physical stimulation versus psychological stimulation ...fidelity of physical stimulation . HUDDLESTON. H.F. and ROLFE, J.M. Behaviouralfactors influencing aircrew response in training and research simulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xue, Peng; School of Public Health, China Medical University, Shenyang 110001; Hou, Yongyong

Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptakemore » (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs{sup 3+} exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes.« less

Bioglass Activated Skin Tissue Engineering Constructs for Wound Healing.

PubMed

Yu, Hongfei; Peng, Jinliang; Xu, Yuhong; Chang, Jiang; Li, Haiyan

2016-01-13

Wound healing is a complicated process, and fibroblast is a major cell type that participates in the process. Recent studies have shown that bioglass (BG) can stimulate fibroblasts to secrete a multitude of growth factors that are critical for wound healing. Therefore, we hypothesize that BG can stimulate fibroblasts to have a higher bioactivity by secreting more bioactive growth factors and proteins as compared to untreated fibroblasts, and we aim to construct a bioactive skin tissue engineering graft for wound healing by using BG activated fibroblast sheet. Thus, the effects of BG on fibroblast behaviors were studied, and the bioactive skin tissue engineering grafts containing BG activated fibroblasts were applied to repair the full skin lesions on nude mouse. Results showed that BG stimulated fibroblasts to express some critical growth factors and important proteins including vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, collagen I, and fibronectin. In vivo results revealed that fibroblasts in the bioactive skin tissue engineering grafts migrated into wound bed, and the migration ability of fibroblasts was stimulated by BG. In addition, the bioactive BG activated fibroblast skin tissue engineering grafts could largely increase the blood vessel formation, enhance the production of collagen I, and stimulate the differentiation of fibroblasts into myofibroblasts in the wound site, which would finally accelerate wound healing. This study demonstrates that the BG activated skin tissue engineering grafts contain more critical growth factors and extracellular matrix proteins that are beneficial for wound healing as compared to untreated fibroblast cell sheets.

The effect of serum on monocyte tissue factor generation.

PubMed

Edwards, R L; Perla, D

1984-09-01

Human monocytes generate the procoagulant tissue factor (MTF) following exposure to a variety of immune stimuli in vitro. The generation of MTF is modified by T cells, lymphokines, and immunoregulatory lipoproteins, and recent studies have shown that MTF can be activated in an immune-specific manner following exposure to antigen. We have examined the role of serum factors in the regulation of MTF generation. Low concentrations (less than 1%) of heat-inactivated normal human serum greatly enhanced MTF generation in cultures of normal peripheral blood mononuclear cells. The stimulatory effect was observed in cultures of both unstimulated cells and cells exposed to bacterial lipopolysaccharide. Stimulation was not observed at high serum concentrations (greater than 10%) and could not be explained by endotoxin contamination or activation of the assay system. Stimulatory activity was present in plasma and BaSO4-adsorbed plasma as well as autologous and allogeneic serum, was not abolished by removal of serum lipoproteins, and did not require the presence of T cells for its expression. Sera from 28 different normal volunteers were screened for stimulatory activity and demonstrated a wide variation in potency. These results suggest that a potent factor is present in sera that enhances the expression of MTF activity in vitro. This factor is distinct from previously described lipoprotein regulators and may play a role in the initiation of coagulation in both normal hemostasis and pathologic states.

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PubMed

Aranha, C; Bordekar, A; Shahani, S

1998-11-01

Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

PFOS induces adipogenesis and glucose uptake in association with activation of Nrf2 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Jialin; Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI 02881; Shimpi, Prajakta

PFOS is a chemical of nearly ubiquitous exposure in humans. Recent studies have associated PFOS exposure to adipose tissue-related effects. The present study was to determine whether PFOS alters the process of adipogenesis and regulates insulin-stimulated glucose uptake in mouse and human preadipocytes. In murine-derived 3T3-L1 preadipocytes, PFOS enhanced hormone-induced differentiation to adipocytes and adipogenic gene expression, increased insulin-stimulated glucose uptake at concentrations ranging from 10 to 100 μM, and enhanced Glucose transporter type 4 and Insulin receptor substrate-1 expression. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), NAD(P)H dehydrogenase, quinone 1 and Glutamate-cysteine ligase, catalytic subunit were significantly induced in 3T3-L1more » cells treated with PFOS, along with a robust induction of Antioxidant Response Element (ARE) reporter in mouse embryonic fibroblasts isolated from ARE-hPAP transgenic mice by PFOS treatment. Chromatin immunoprecipitation assays further illustrated that PFOS increased Nrf2 binding to ARE sites in mouse Nqo1 promoter, suggesting that PFOS activated Nrf2 signaling in murine-derived preadipocytes. Additionally, PFOS administration in mice (100 μg/kg/day) induced adipogenic gene expression and activated Nrf2 signaling in epididymal white adipose tissue. Moreover, the treatment on human visceral preadipocytes illustrated that PFOS (5 and 50 μM) promoted adipogenesis and increased cellular lipid accumulation. It was observed that PFOS increased Nrf2 binding to ARE sites in association with Nrf2 signaling activation, induction of Peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α expression, and increased adipogenesis. This study points to a potential role of PFOS in dysregulation of adipose tissue expandability, and warrants further investigations on the adverse effects of persistent pollutants on human health. - Highlights: • PFOS induces adipogenesis in association with increased Pparγ and Cebpα mRNA expression. • PFOS increases ARE binding activity and activates Nrf2 signaling. • PFOS increases insulin-stimulated glucose uptake.« less

Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

PubMed

Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

2015-01-30

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

Tissue Factor Inflammatory Response Regulated by Promoter Genotype and p38 MAPK in Neonatal vs. Adult Microvascular Endothelial Cells

PubMed Central

Buzby, Jeffrey S.; Williams, Shirley A.; Imfeld, Karen L.; Kunicki, Thomas J.; Nugent, Diane J.

2014-01-01

Objective and design Variable tissue factor (TF) expression by human microvascular endothelial cells (HMVEC) may be regulated by two promoter haplotypes, distinguished by an 18 base pair deletion (D) or insertion (I) at -1208. We sought to determine the relationship between these haplotypes and interleukin-1 (IL-1α)-induced TF expression in neonatal versus adult HMVEC. Results IL-1-stimulated TF mRNA, protein, and activity were significantly higher in neonatal compared to adult D/D donors. IL-1-stimulated HMVEC from neonatal D/D donors expressed 3-fold higher levels of TF mRNA, 2-fold higher TF protein, and 4-fold increased TF activity compared to HMVEC from adult D/D donors. These results indicate that homozygosity for the D haplotype is characterized by increased response to IL-1 in neonates but not adults. IL-1 induced increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), which was significantly greater in neonatal compared to adult HMVEC. Moreover, inhibition of the p38 MAPK pathway reduced IL-1-stimulated TF mRNA expression in D/D neonatal but not adult HMVEC. Conclusions Up-regulation of D/D neonatal HMVEC TF expression by IL-1 is mediated through the p38 MAPK pathway. This heightened response of D/D neonatal HMVEC to inflammatory stimuli may contribute to increased microvascular coagulopathies in susceptible newborn infants. PMID:24385191

Inhibitory effects of tocopherols on expression of the cyclooxygenase-2 gene in RAW264.7 cells stimulated by lipopolysaccharide, tumor necrosis factor-α or Porphyromonas gingivalis fimbriae.

PubMed

Murakami, Yukio; Kawata, Akifumi; Koh, Teho; Seki, Yuya; Tamura, Seiko; Katayama, Tadashi; Fujisawa, Seiichiro

2013-01-01

Tocopherols, which include α-, β-, γ-, and δ-tocopherol, protect cells against harmful free radicals and play an important role in preventing many human diseases such as cancer, inflammatory disorders, and ageing itself. However, the causal relationships between periodontal or oral chronic diseases and tocopherols have not been sufficiently studied. The present study investigated the inhibitory effects of these compounds on the expression of cyclooxygenase-2 (COX2) mRNA in RAW264.7 cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor-α (TNFα) or fimbriae of Poryphyromonas gingivalis (Pg), an oral anaerobe. The cytotoxicity (EC₅₀) of tocopherols toward RAW cells was determined using a cell counting kit (CCK-8). The regulatory effect of these compounds on the expression of COX2 mRNA stimulated with LPS, TNFα or Pg fimbriae was investigated using real-time polymerase chain reaction (PCR). Each tocopherol had similarly low cytotoxicity. COX2 gene expression in RAW cells after exposure to the three different macrophage activators was inhibited by the tocopherols (p<0.01). Compared to α-tocopherol, β-, γ- and δ-tocopherol exhibited greater inhibitory effects (p<0.05). Tocopherols exhibit anti-inflammatory activity, and β-, γ- and δ-tocopherol have particularly more potent anti-inflammatory activity than α-tocopherol. Tocopherols may have potential utility for prevention of periodontal and chronic oral diseases.

Early tumor growth in metastatic organs influenced by the microenvironment is an important factor which provides organ specificity of colon cancer metastasis.

PubMed

Hara, Y; Ogata, Y; Shirouzu, K

2000-12-01

We have previously demonstrated that liver metastases in nude mice and lung metastases in nude rats occurred specifically, when KM12SM human colon carcinoma cells were inoculated orthotopically into the cecal wall of nude mice and rats. To clarify the relationship between the tumor growth potential in the metastatic organs and the metastatic organ preference in these two metastatic models, we have evaluated the in vitro cell growth activities affected by the organ conditioned medium (CM) from the liver and lung, and the in vivo growth activities of the ectopic implanted tumors in the liver and lung. The tumorigenicity of the ectopic implanted tumors was 100% in mouse liver, 33% in rat liver, 50% in mouse lung, and 75% in rat lung. The crude liver CM of the animals showed inhibitory activities for KM12SM cell growth in a dosage-dependent manner, and the crude lung CM stimulated KM12SM cell growth. The liver CM of nude mice inhibited the KM12SM cell growth more strongly compared with the CM of nude rats, and the lung CM of nude rats was more strongly stimulated compared with the CM of nude mice. The liver CM of nude mice had non-heparin binding factors, which stimulated or inhibited KM12SM cell growth, in a molecular weight range of 50 to 100 kDa. By contrast, the liver CM of nude rats showed no growth stimulating activity for KM12SM cells. These results suggest that the metastatic organ specificity of KM12SM cells may depend on the early tumor growth influenced by the microenvironment in metastatic organs.

Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

PubMed Central

Brighenti, E; Calabrese, C; Liguori, G; Giannone, F A; Trerè, D; Montanaro, L; Derenzini, M

2014-01-01

Chronic inflammation is an established risk factor for the onset of cancer, and the inflammatory cytokine IL-6 has a role in tumorigenesis by enhancing proliferation and hindering apoptosis. As factors stimulating proliferation also downregulate p53 expression by enhancing ribosome biogenesis, we hypothesized that IL-6 may cause similar changes in inflamed tissues, thus activating a mechanism that favors neoplastic transformation. Here, we showed that IL-6 downregulated the expression and activity of p53 in transformed and untransformed human cell lines. This was the consequence of IL-6-dependent stimulation of c-MYC mRNA translation, which was responsible for the upregulation of rRNA transcription. The enhanced rRNA transcription stimulated the MDM2-mediated proteasomal degradation of p53, by reducing the availability of ribosome proteins for MDM2 binding. The p53 downregulation induced the acquisition of cellular phenotypic changes characteristic of epithelial–mesenchymal transition, such as a reduced level of E-cadherin expression, increased cell invasiveness and a decreased response to cytotoxic stresses. We found that these changes also occurred in colon epithelial cells of patients with ulcerative colitis, a very representative example of chronic inflammation at high risk for tumor development. Histochemical and immunohistochemical analysis of colon biopsy samples showed an upregulation of ribosome biogenesis, a reduced expression of p53, together with a focal reduction or absence of E-cadherin expression in chronic colitis in comparison with normal mucosa samples. These changes disappeared after treatment with anti-inflammatory drugs. Taken together, the present results highlight a new mechanism that may link chronic inflammation to cancer, based on p53 downregulation, which is activated by the enhancement of rRNA transcription upon IL-6 exposure. PMID:24531714

Beta-Glucan Activated Human B-Lymphocytes Participate in Innate Immune Responses by Releasing Pro-inflammatory Cytokines and Stimulating Neutrophil Chemotaxis

PubMed Central

Ali, Mohamed F.; Driscoll, Christopher B.; Walters, Paula R.; Limper, Andrew H.; Carmona, Eva M.

2015-01-01

B-lymphocytes play an essential regulatory role in the adaptive immune response through antibody production during infection. A less known function of B-lymphocytes is their ability to respond directly to infectious antigens through stimulation of pattern recognition receptors expressed on their surfaces. β-glucans are carbohydrates present in the cell wall of many pathogenic fungi that can be detected in the peripheral blood of patients during infection. They have been shown to participate in the innate inflammatory response as they can directly activate peripheral macrophages and dendritic cells. However, their effect as direct stimulators of B-lymphocytes has not been yet fully elucidated. The aim of this study was to examine the molecular mechanisms and cytokine profiles generated following β-glucan stimulation of B-lymphocytes, compared with the well-established TLR-9 agonist CpG-oligodeoxynucleotide (CpG) and study the participation of β-glucan stimulated B-cells in the innate immune response. Herein, we demonstrate that β-glucan activated B-lymphocytes upregulate pro-inflammatory cytokines (TNFα, IL-6 and IL-8). Interestingly, β-glucan, unlike CpG, had no effect on B-lymphocyte proliferation or IgM production. When compared with CpG (TLR9 agonist), β-glucan-activated cells secreted significantly higher levels of IL-8. Furthermore, IL-8 secretion was partially mediated by Dectin-1 and required SYK, MAPKs and the transcription factors NF-κB and AP-1. Moreover, we observed that conditioned media from β-glucan stimulated B-lymphocytes elicited neutrophil chemotaxis. These studies suggest that β-glucan activated B-lymphocytes have an important and novel role in fungal innate immune responses. PMID:26519534

Rapamycin has suppressive and stimulatory effects on human plasmacytoid dendritic cell functions

PubMed Central

Boor, P P C; Metselaar, H J; Mancham, S; van der Laan, L J W; Kwekkeboom, J

2013-01-01

Plasmacytoid dendritic cells (PDC) are involved in innate immunity by interferon (IFN)-α production, and in adaptive immunity by stimulating T cells and inducing generation of regulatory T cells (Treg). In this study we studied the effects of mammalian target of rapamycin (mTOR) inhibition by rapamycin, a commonly used immunosuppressive and anti-cancer drug, on innate and adaptive immune functions of human PDC. A clinically relevant concentration of rapamycin inhibited Toll-like receptor (TLR)-7-induced IFN-α secretion potently (−64%) but TLR-9-induced IFN-α secretion only slightly (−20%), while the same concentration suppressed proinflammatory cytokine production by TLR-7-activated and TLR-9-activated PDC with similar efficacy. Rapamycin inhibited the ability of both TLR-7-activated and TLR-9-activated PDC to stimulate production of IFN-γ and interleukin (IL)-10 by allogeneic T cells. Surprisingly, mTOR-inhibition enhanced the capacity of TLR-7-activated PDC to stimulate naive and memory T helper cell proliferation, which was caused by rapamycin-induced up-regulation of CD80 expression on PDC. Finally, rapamycin treatment of TLR-7-activated PDC enhanced their capacity to induce CD4+forkhead box protein 3 (FoxP3)+ regulatory T cells, but did not affect the generation of suppressive CD8+CD38+lymphocyte activation gene (LAG)-3+ Treg. In general, rapamycin inhibits innate and adaptive immune functions of TLR-stimulated human PDC, but enhances the ability of TLR-7-stimulated PDC to stimulate CD4+ T cell proliferation and induce CD4+FoxP3+ regulatory T cell generation. PMID:23968562

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

PubMed Central

Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

2015-01-01

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

Immunomodulatory properties of human periodontal ligament stem cells.

PubMed

Wada, Naohisa; Menicanin, Danijela; Shi, Songtao; Bartold, P Mark; Gronthos, Stan

2009-06-01

Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell-based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non-cell contact dependent suppression of PBMNC proliferation in co-cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN-gamma partially suppressed PBMNC proliferation when compared to CMs without IFN-gamma stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF-beta1, hepatocyte growth factor (HGF) and indoleamine 2, 3-dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN-gamma. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667-676, 2009. (c) 2009 Wiley-Liss, Inc.

Long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer.

PubMed

Ronca, Roberto; Alessi, Patrizia; Coltrini, Daniela; Di Salle, Emanuela; Giacomini, Arianna; Leali, Daria; Corsini, Michela; Belleri, Mirella; Tobia, Chiara; Garlanda, Cecilia; Bonomi, Elisa; Tardanico, Regina; Vermi, William; Presta, Marco

2013-06-01

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes

PubMed Central

Köllisch, Gabriele; Kalali, Behnam Naderi; Voelcker, Verena; Wallich, Reinhard; Behrendt, Heidrun; Ring, Johannes; Bauer, Stefan; Jakob, Thilo; Mempel, Martin; Ollert, Markus

2005-01-01

Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-κB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1–10 expression profile. First, reverse transcription–polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1–10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-κB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-κB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin. PMID:15804290

Evidence for the separate human T-lymphocyte subpopulations that collaborate with autologous monocyte/macrophages in the elaboration of colony-stimulating activity and those that suppress this collaboration.

PubMed

Verma, D S; Johnston, D A; McCredie, K B

1983-11-01

We investigated the interaction of monocyte/macrophages and autologous T lymphocytes in the methanol extraction residue (MER) of BCG-induced production of granulocyte-macrophage colony-stimulating activity (CSA). Coincubation of monocyte/macrophages and T lymphocytes at a 1:3 ratio produces an optimum collaboration; a change to a 1:9 ratio diminished this collaboration. Coincubation of monocyte/macrophages and T lymphocytes primed with lithium carbonate (2 meq/liter) for 40 hr synergistically increased CSA elaboration and prevented the decline in CSA noted for the 1:9 monocyte/macrophage: T lymphocyte ratio. In contrast, concanavalin-A-primed T lymphocytes did not enhance CSA elaboration at any monocyte/macrophage:T lymphocyte ratio except, occasionally, at 1:9. However, this was overcome if the T lymphocytes were primed with both concanavalin-A and lithium carbonate before their coincubation with monocyte/macrophages. Further cell-mixing experiments revealed that concanavalin-A-primed T lymphocytes contained a subpopulation that suppressed monocyte/macrophage and T-lymphocyte collaboration. Activation of suppressor T lymphocytes could be effectively prevented by lithium carbonate and, in a dose-dependent manner, by irradiation. Also, suppressor T lymphocytes not only diminished the elaboration of colony-stimulating factor(s), but also elaborated an inhibitor of granulocyte-macrophage colony-forming cells. We further demonstrated that the respective hemopoietic helper and suppressor T-lymphocyte activities could be enriched with OKT8- (or OKT4+) and OKT8+ subpopulations.

Posterior Cricoarytenoid Muscle Dynamics in Canines and Humans

PubMed Central

Chhetri, Dinesh K.; Neubauer, Juergen; Sofer, Elazar

2015-01-01

Objective The posterior cricoarytenoid (PCA) muscle is the sole abductor of the glottis and serves important functions during respiration, phonation, cough, and sniff. The present study examines vocal fold abduction dynamics during PCA muscle activation. Study Design Basic science study using an in vivo canine model and human subjects. Methods In four canines and five healthy humans vocal fold abduction time was measured using high speed video recording. In the canines, PCA muscle activation was achieved using graded stimulation of the PCA nerve branch. The human subjects performed coughing and sniffing tasks. High speed video and audio signals were concurrently recorded. Results In the canines the vocal fold moved posteriorly, laterally, and superiorly during abduction. Average time to reach 10%, 50% and 90% abduction was 23, 50, and 100 ms with low stimulation, 24, 58, and 129 ms with medium stimulation, and 21, 49, and 117 ms with high level stimulation. In the humans, 100% abduction times for coughing and sniffing tasks were 79 and 193 ms, respectively. Conclusion The PCA abduction times in canines are within the range in humans. The results also further support the notion that PCA muscles are fully active during cough. Level of Evidence N/A (Animal studies and basic research) PMID:24781959

R/S analysis of reaction time in Neuron Type Test for human activity in civil aviation

NASA Astrophysics Data System (ADS)

Zhang, Hong-Yan; Kang, Ming-Cui; Li, Jing-Qiang; Liu, Hai-Tao

2017-03-01

Human factors become the most serious problem leading to accidents of civil aviation, which stimulates the design and analysis of Neuron Type Test (NTT) system to explore the intrinsic properties and patterns behind the behaviors of professionals and students in civil aviation. In the experiment, normal practitioners' reaction time sequences, collected from NTT, exhibit log-normal distribution approximately. We apply the χ2 test to compute the goodness-of-fit by transforming the time sequence with Box-Cox transformation to cluster practitioners. The long-term correlation of different individual practitioner's time sequence is represented by the Hurst exponent via Rescaled Range Analysis, also named by Range/Standard deviation (R/S) Analysis. The different Hurst exponent suggests the existence of different collective behavior and different intrinsic patterns of human factors in civil aviation.

Hyperglycemia induces mixed M1/M2 cytokine profile in primary human monocyte-derived macrophages.

PubMed

Moganti, Kondaiah; Li, Feng; Schmuttermaier, Christina; Riemann, Sarah; Klüter, Harald; Gratchev, Alexei; Harmsen, Martin C; Kzhyshkowska, Julia

2017-10-01

Hyperglycaemia is a key factor in diabetic pathology. Macrophages are essential regulators of inflammation which can be classified into two major vectors of polarisation: classically activated macrophages (M1) and alternatively activated macrophages (M2). Both types of macrophages play a role in diabetes, where M1 and M2-produced cytokines can have detrimental effects in development of diabetes-associated inflammation and diabetic vascular complications. However, the effect of hyperglycaemia on differentiation and programming of primary human macrophages was not systematically studied. We established a unique model to assess the influence of hyperglycaemia on M1 and M2 differentiation based on primary human monocyte-derived macrophages. The effects of hyperglycaemia on the gene expression and secretion of prototype M1 cytokines TNF-alpha and IL-1beta, and prototype M2 cytokines IL-1Ra and CCL18 were quantified by RT-PCR and ELISA. Hyperglycaemia stimulated production of TNF-alpha, IL-1beta and IL-1Ra during macrophage differentiation. The effect of hyperglycaemia on TNF-alpha was acute, while the stimulating effect on IL-1beta and IL-1Ra was constitutive. Expression of CCL18 was supressed in M2 macrophages by hyperglycaemia. However the secreted levels remained to be biologically significant. Our data indicate that hyperglycaemia itself, without additional metabolic factors induces mixed M1/M2 cytokine profile that can support of diabetes-associated inflammation and development of vascular complications. Copyright © 2016 Elsevier GmbH. All rights reserved.

High Definition Transcranial Direct Current Stimulation Induces Both Acute and Persistent Changes in Broadband Cortical Synchronization: a Simultaneous tDCS-EEG Study

PubMed Central

Roy, Abhrajeet; Baxter, Bryan

2014-01-01

The goal of this study was to develop methods for simultaneously acquiring electrophysiological data during high definition transcranial direct current stimulation (tDCS) using high resolution electroencephalography (EEG). Previous studies have pointed to the after effects of tDCS on both motor and cognitive performance, and there appears to be potential for using tDCS in a variety of clinical applications. However, little is known about the real-time effects of tDCS on rhythmic cortical activity in humans due to the technical challenges of simultaneously obtaining electrophysiological data during ongoing stimulation. Furthermore, the mechanisms of action of tDCS in humans are not well understood. We have conducted a simultaneous tDCS-EEG study in a group of healthy human subjects. Significant acute and persistent changes in spontaneous neural activity and event related synchronization (ERS) were observed during and after the application of high definition tDCS over the left sensorimotor cortex. Both anodal and cathodal stimulation resulted in acute global changes in broadband cortical activity which were significantly different than the changes observed in response to sham stimulation. For the group of 8 subjects studied, broadband individual changes in spontaneous activity during stimulation were apparent both locally and globally. In addition, we found that high definition tDCS of the left sensorimotor cortex can induce significant ipsilateral and contralateral changes in event related desynchronization (ERD) and ERS during motor imagination following the end of the stimulation period. Overall, our results demonstrate the feasibility of acquiring high resolution EEG during high definition tDCS and provide evidence that tDCS in humans directly modulates rhythmic cortical synchronization during and after its administration. PMID:24956615

TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils.

PubMed

Yokose, Kohei; Sato, Shuzo; Asano, Tomoyuki; Yashiro, Makiko; Kobayashi, Hiroko; Watanabe, Hiroshi; Suzuki, Eiji; Sato, Chikako; Kozuru, Hideko; Yatsuhashi, Hiroshi; Migita, Kiyoshi

2018-05-01

Monosodium urate (MSU) has been shown to promote interleukin-1β (IL-1β) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1β induction in human neutrophils. Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1β, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1β mRNA expressions in human neutrophils were analyzed by real-time PCR method. TNF-α stimulation induced pro-IL-1β mRNA expression; however, MSU stimulation did not induce pro-IL-1β mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1β secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1β and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation. Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

Neutral sphingomyelinase-3 mediates TNF-stimulated oxidant activity in skeletal muscle.

PubMed

Moylan, Jennifer S; Smith, Jeffrey D; Wolf Horrell, Erin M; McLean, Julie B; Deevska, Gergana M; Bonnell, Mark R; Nikolova-Karakashian, Mariana N; Reid, Michael B

2014-01-01

Sphingolipid and oxidant signaling affect glucose uptake, atrophy, and force production of skeletal muscle similarly and both are stimulated by tumor necrosis factor (TNF), suggesting a connection between systems. Sphingolipid signaling is initiated by neutral sphingomyelinase (nSMase), a family of agonist-activated effector enzymes. Northern blot analyses suggest that nSMase3 may be a striated muscle-specific nSMase. The present study tested the hypothesis that nSMase3 protein is expressed in skeletal muscle and functions to regulate TNF-stimulated oxidant production. We demonstrate constitutive nSMase activity in skeletal muscles of healthy mice and humans and in differentiated C2C12 myotubes. nSMase3 (Smpd4 gene) mRNA is highly expressed in muscle. An nSMase3 protein doublet (88 and 85 kD) is derived from alternative mRNA splicing of exon 11. The proteins partition differently. The full-length 88 kD isoform (nSMase3a) fractionates with membrane proteins that are resistant to detergent extraction; the 85 kD isoform lacking exon 11 (nSMase3b) is more readily extracted and fractionates with detergent soluble membrane proteins; neither variant is detected in the cytosol. By immunofluorescence microscopy, nSMase3 resides in both internal and sarcolemmal membranes. Finally, myotube nSMase activity and cytosolic oxidant activity are stimulated by TNF. Both if these responses are inhibited by nSMase3 knockdown. These findings identify nSMase3 as an intermediate that links TNF receptor activation, sphingolipid signaling, and skeletal muscle oxidant production. Our data show that nSMase3 acts as a signaling nSMase in skeletal muscle that is essential for TNF-stimulated oxidant activity.

Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells.

PubMed

Fujino, Hiromichi; Seira, Naofumi; Kurata, Naoki; Araki, Yumi; Nakamura, Hiroyuki; Regan, John W; Murayama, Toshihiko

2015-12-05

Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gαi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gαs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gαs-protein/cAMP/PKA pathway in development of colon cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.

PubMed

Ismail, Heba M; Yamamoto, Kazuhiro; Vincent, Tonia L; Nagase, Hideaki; Troeberg, Linda; Saklatvala, Jeremy

2015-07-01

Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes. We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting. ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1. The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its endocytosis, which may be reduced upon IL-1 stimulation because of LRP-1 shedding. © 2015, American College of Rheumatology.

High-resolution measurement of electrically-evoked vagus nerve activity in the anesthetized dog

NASA Astrophysics Data System (ADS)

Yoo, Paul B.; Lubock, Nathan B.; Hincapie, Juan G.; Ruble, Stephen B.; Hamann, Jason J.; Grill, Warren M.

2013-04-01

Objective. Not fully understanding the type of axons activated during vagus nerve stimulation (VNS) is one of several factors that limit the clinical efficacy of VNS therapies. The main goal of this study was to characterize the electrical recruitment of both myelinated and unmyelinated fibers within the cervical vagus nerve. Approach. In anesthetized dogs, recording nerve cuff electrodes were implanted on the vagus nerve following surgical excision of the epineurium. Both the vagal electroneurogram (ENG) and laryngeal muscle activity were recorded in response to stimulation of the right vagus nerve. Main results. Desheathing the nerve significantly increased the signal-to-noise ratio of the ENG by 1.2 to 9.9 dB, depending on the nerve fiber type. Repeated VNS following nerve transection or neuromuscular block (1) enabled the characterization of A-fibers, two sub-types of B-fibers, and unmyelinated C-fibers, (2) confirmed the absence of stimulation-evoked reflex compound nerve action potentials in both the ipsilateral and contralateral vagus nerves, and (3) provided evidence of stimulus spillover into muscle tissue surrounding the stimulating electrode. Significance. Given the anatomical similarities between the canine and human vagus nerves, the results of this study provide a template for better understanding the nerve fiber recruitment patterns associated with VNS therapies.

Cyclic strain alters the expression and release of angiogenic factors by human tendon cells.

PubMed

Mousavizadeh, Rouhollah; Khosravi, Shahram; Behzad, Hayedeh; McCormack, Robert G; Duronio, Vincent; Scott, Alex

2014-01-01

Angiogenesis is associated with the tissue changes underlying chronic overuse tendinopathy. We hypothesized that repetitive, cyclic loading of human tendon cells would lead to increased expression and activity of angiogenic factors. We subjected isolated human tendon cells to overuse tensile loading using an in vitro model (1 Hz, 10% equibiaxial strain). We found that mechanically stimulated human tendon cells released factors that promoted in vitro proliferation and tube formation by human umbilical vein endothelial cells (HUVEC). In response to cyclic strain, there was a transient increase in the expression of several angiogenic genes including ANGPTL4, FGF-2, COX-2, SPHK1, TGF-alpha, VEGF-A and VEGF-C, with no change in anti-angiogenic genes (BAI1, SERPINF1, THBS1 and 2, TIMP1-3). Cyclic strain also resulted in the extracellular release of ANGPTL4 protein by tendon cells. Our study is the first report demonstrating the induction of ANGPTL4 mRNA and release of ANGPTL4 protein in response to cyclic strain. Tenocytes may contribute to the upregulation of angiogenesis during the development of overuse tendinopathy.

Cyclic Strain Alters the Expression and Release of Angiogenic Factors by Human Tendon Cells

PubMed Central

Mousavizadeh, Rouhollah; Khosravi, Shahram; Behzad, Hayedeh; McCormack, Robert G.; Duronio, Vincent; Scott, Alex

2014-01-01

Angiogenesis is associated with the tissue changes underlying chronic overuse tendinopathy. We hypothesized that repetitive, cyclic loading of human tendon cells would lead to increased expression and activity of angiogenic factors. We subjected isolated human tendon cells to overuse tensile loading using an in vitro model (1 Hz, 10% equibiaxial strain). We found that mechanically stimulated human tendon cells released factors that promoted in vitro proliferation and tube formation by human umbilical vein endothelial cells (HUVEC). In response to cyclic strain, there was a transient increase in the expression of several angiogenic genes including ANGPTL4, FGF-2, COX-2, SPHK1, TGF-alpha, VEGF-A and VEGF-C, with no change in anti-angiogenic genes (BAI1, SERPINF1, THBS1 and 2, TIMP1-3). Cyclic strain also resulted in the extracellular release of ANGPTL4 protein by tendon cells. Our study is the first report demonstrating the induction of ANGPTL4 mRNA and release of ANGPTL4 protein in response to cyclic strain. Tenocytes may contribute to the upregulation of angiogenesis during the development of overuse tendinopathy. PMID:24824595

TAM receptor-dependent regulation of SOCS3 and MAPKs contributes to pro-inflammatory cytokine downregulation following chronic NOD2 stimulation of human macrophages1

PubMed Central

Zheng, Shasha; Hedl, Matija; Abraham, Clara

2014-01-01

Microbial-induced cytokine regulation is critical to intestinal immune homeostasis. Acute stimulation of NOD2, the Crohn’s disease-associated sensor of bacterial peptidoglycan, induces cytokines. However, cytokines are attenuated after chronic NOD2 and pattern recognition receptor (PRR) stimulation of macrophages; similar attenuation is observed in intestinal macrophages. The role of Tyro3, Axl and Mer (TAM) receptors in regulating chronic PRR stimulation and NOD2-induced outcomes has not been examined. Moreover, TAM receptors have been relatively less investigated in human macrophages. Whereas TAM receptors did not downregulate acute NOD2-induced cytokines in primary human macrophages, they were essential for downregulating signaling and pro-inflammatory cytokine secretion after chronic NOD2 and TLR4 stimulation. Axl and Mer were similarly required in mice for cytokine downregulation after chronic NOD2 stimulation in vivo and in intestinal tissues. Consistently, TAM expression was increased in human intestinal myeloid-derived cells. Chronic NOD2 stimulation led to IL-10- and TGFβ-dependent TAM upregulation in human macrophages, which in turn, upregulated SOCS3 expression. Restoring SOCS3 expression under TAM knockdown conditions restored chronic NOD2-mediated pro-inflammatory cytokine downregulation. In contrast to the upregulated pro-inflammatory cytokines, attenuated IL-10 secretion was maintained in TAM-deficient macrophages upon chronic NOD2 stimulation. The level of MAPK activation in TAM-deficient macrophages after chronic NOD2 stimulation was insufficient to upregulate IL-10 secretion; however, full restoration of MAPK activation under these conditions restored c-Fos, c-Jun, MAFK and PU.1 binding to the IL-10 promoter and IL-10 secretion. Therefore, TAM receptors are critical for downregulating pro-inflammatory cytokines under the chronic NOD2 stimulation conditions observed in the intestinal environment. PMID:25567680

Biological Mechanisms Underlying the Ultraviolet Radiation-Induced Formation of Skin Wrinkling and Sagging II: Over-Expression of Neprilysin Plays an Essential Role

PubMed Central

Imokawa, Genji; Nakajima, Hiroaki; Ishida, Koichi

2015-01-01

Our previous studies strongly indicated that the up-regulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. Fortunately, we succeeded in identifying human skin fibroblast-derived elastase as a previously known enzyme, neprilysin or neutral endopeptidase (NEP). We have also characterized epithelial-mesenchymal paracrine cytokine interactions between UVB-exposed-keratinocytes and dermal fibroblasts and found that interleukin-1α and granulocyte macrophage colony stimulatory factor (GM-CSF) are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by fibroblasts. On the other hand, direct UVA exposure of human fibroblasts significantly stimulates the secretion of IL-6 and also elicits a significant increase in the gene expression of matrix metallo-protease(MMP)-1 as well as neprilysin (to a lesser extent), which is followed by distinct increases in their protein and enzymatic activity levels. Direct UVA exposure of human keratinocytes also stimulates the secretion of IL-6, IL-8 and GM-CSF but not of IL-1 and endothelin-1. These findings suggest that GM-CSF secreted by UVA-exposed keratinocytes as well as IL-6 secreted by UVA-exposed dermal fibroblasts play important and additional roles in UVA-induced sagging and wrinkling by up-regulation of neprilysin and MMP-1, respectively, in dermal fibroblasts. PMID:25856676

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

PubMed

Akuthota, Praveen; Melo, Rossana C N; Spencer, Lisa A; Weller, Peter F

2012-02-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

Spike-timing-dependent plasticity in the human dorso-lateral prefrontal cortex.

PubMed

Casula, Elias Paolo; Pellicciari, Maria Concetta; Picazio, Silvia; Caltagirone, Carlo; Koch, Giacomo

2016-12-01

Changes in the synaptic strength of neural connections are induced by repeated coupling of activity of interconnected neurons with precise timing, a phenomenon known as spike-timing-dependent plasticity (STDP). It is debated if this mechanism exists in large-scale cortical networks in humans. We combined transcranial magnetic stimulation (TMS) with concurrent electroencephalography (EEG) to directly investigate the effects of two paired associative stimulation (PAS) protocols (fronto-parietal and parieto-frontal) of pre and post-synaptic inputs within the human fronto-parietal network. We found evidence that the dorsolateral prefrontal cortex (DLPFC) has the potential to form robust STDP. Long-term potentiation/depression of TMS-evoked cortical activity is prompted after that DLPFC stimulation is followed/preceded by posterior parietal stimulation. Such bidirectional changes are paralleled by sustained increase/decrease of high-frequency oscillatory activity, likely reflecting STDP responsivity. The current findings could be important to drive plasticity of damaged cortical circuits in patients with cognitive or psychiatric disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parvinen, M.; Soeder, O.M.; Mali, P.

Levels of rat testicular interleukin-1-like factor (tIL-1) have been shown to correlate with DNA synthetic activity during the cycle of the rat seminiferous epithelium, suggesting its role as a spermatogonial or meiotic growth factor. To explore this further, a new in vitro model system was developed. Rat seminiferous tubule segments from stages I, V, VIIa, and VIII-IX of the cycle were isolated by transillumination-assisted microdissection, cultured in chemically defined serum-free medium supplemented with human recombinant IL-1 {alpha}, and labeled with (3H)thymidine. During incubation, spontaneous progression of spermatogenesis was noted. Inactive stage VIIa tubule segments differentiated to stage VIII and initiatedmore » DNA synthesis, and concomitantly started to secrete IL-1-like factor. DNA synthesis of stages VIII-IX ceased through differentiation of spermatocytes to leptotene-zygotene (stages XII-XIII of the cycle). IL-1 {alpha} stimulated DNA synthesis significantly in spermatogonia of stage I. Meiotic DNA synthesis at stage VIIa was stimulated (48 h/34 C) and maintained at stages VIII-IX (48 h/34 C). IL-1 {alpha} seems to act as a regulator of spermatogenic DNA synthesis in both mitotic and meiotic phases. It has mainly stimulating and maintaining effects, but it may also be inhibitory under certain conditions.« less

Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

PubMed

Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

2015-01-01

We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

Effect of a high and low dose of caffeine on human lymphocyte activation in response to antigen stimulation.

PubMed

Dulson, Deborah K; Bishop, Nicolette C

2016-02-01

This study investigated the effect of caffeine on antigen-stimulated lymphocyte activation. Six males rested for 3.5 h after ingesting 0 (PLA), 2, or 6 (6CAF) mg·kg(-1) body mass of caffeine. The number of antigen-stimulated NK CD69(+) cells increased in 6CAF at 1 h compared with PLA (P = 0.021). Caffeine did not influence the number of antigen-stimulated CD69(+) T cells or the geometric mean fluorescence intensity expression of CD69 on antigen-stimulated lymphocytes, suggesting caffeine has little effect on antigen-stimulated lymphocyte activation.

The peripheral clock regulates human pigmentation.

PubMed

Hardman, Jonathan A; Tobin, Desmond J; Haslam, Iain S; Farjo, Nilofer; Farjo, Bessam; Al-Nuaimi, Yusur; Grimaldi, Benedetto; Paus, Ralf

2015-04-01

Although the regulation of pigmentation is well characterized, it remains unclear whether cell-autonomous controls regulate the cyclic on-off switching of pigmentation in the hair follicle (HF). As human HFs and epidermal melanocytes express clock genes and proteins, and given that core clock genes (PER1, BMAL1) modulate human HF cycling, we investigated whether peripheral clock activity influences human HF pigmentation. We found that silencing BMAL1 or PER1 in human HFs increased HF melanin content. Furthermore, tyrosinase expression and activity, as well as TYRP1 and TYRP2 mRNA levels, gp100 protein expression, melanocyte dendricity, and the number gp100+ HF melanocytes, were all significantly increased in BMAL1 and/or PER1-silenced HFs. BMAL1 or PER1 silencing also increased epidermal melanin content, gp100 protein expression, and tyrosinase activity in human skin. These effects reflect direct modulation of melanocytes, as BMAL1 and/or PER1 silencing in isolated melanocytes increased tyrosinase activity and TYRP1/2 expression. Mechanistically, BMAL1 knockdown reduces PER1 transcription, and PER1 silencing induces phosphorylation of the master regulator of melanogenesis, microphthalmia-associated transcription factor, thus stimulating human melanogenesis and melanocyte activity in situ and in vitro. Therefore, the molecular clock operates as a cell-autonomous modulator of human pigmentation and may be targeted for future therapeutic strategies.

Acemannan sponges stimulate alveolar bone, cementum and periodontal ligament regeneration in a canine class II furcation defect model.

PubMed

Chantarawaratit, P; Sangvanich, P; Banlunara, W; Soontornvipart, K; Thunyakitpisal, P

2014-04-01

Periodontal disease is a common infectious disease, found worldwide, causing the destruction of the periodontium. The periodontium is a complex structure composed of both soft and hard tissues, thus an agent applied to regenerate the periodontium must be able to stimulate periodontal ligament, cementum and alveolar bone regeneration. Recent studies demonstrated that acemannan, a polysaccharide extracted from Aloe vera gel, stimulated both soft and hard tissue healing. This study investigated effect of acemannan as a bioactive molecule and scaffold for periodontal tissue regeneration. Primary human periodontal ligament cells were treated with acemannan in vitro. New DNA synthesis, expression of growth/differentiation factor 5 and runt-related transcription factor 2, expression of vascular endothelial growth factor, bone morphogenetic protein-2 and type I collagen, alkaline phosphatase activity, and mineralized nodule formation were determined using [(3)H]-thymidine incorporation, reverse transcription-polymerase chain reaction, enzyme-linked immunoabsorbent assay, biochemical assay and alizarin red staining, respectively. In our in vivo study, premolar class II furcation defects were made in four mongrel dogs. Acemannan sponges were applied into the defects. Untreated defects were used as a negative control group. The amount of new bone, cementum and periodontal ligament formation were evaluated 30 and 60 d after the operation. Acemannan significantly increased periodontal ligament cell proliferation, upregulation of growth/differentiation factor 5, runt-related transcription factor 2, vascular endothelial growth factor, bone morphogenetic protein 2, type I collagen and alkaline phosphatase activity, and mineral deposition as compared with the untreated control group in vitro. Moreover, acemannan significantly accelerated new alveolar bone, cementum and periodontal ligament formation in class II furcation defects. Our data suggest that acemannan could be a candidate biomolecule for periodontal tissue regeneration. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The G Protein-Coupled Bile Acid Receptor, TGR5, Stimulates Gallbladder Filling

PubMed Central

Li, Tingting; Holmstrom, Sam R.; Kir, Serkan; Umetani, Michihisa; Schmidt, Daniel R.

2011-01-01

TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5−/− mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of fibroblast growth factor 15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease. PMID:21454404

Interleukin 6 stimulates hepatic glucose release from prelabeled glycogen pools

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritchie, D.G.

1990-01-01

Cytokines, derived from a wide variety of cell types, are now believed to initiate many of the physiological responses accompanying the inflammatory phase that follows either Gram-negative septicemia or thermal injury. Because hypoglycemia (after endotoxic challenge) and hyperglycemia (after thermal injury) represent well-characterized responses to these injuries, we sought to determine whether hepatic glycogen metabolism could be altered by specific cytokines. Cultured adult rat hepatocytes were prelabeled with ({sup 14}C)glucose for 24 h, a procedure that resulted in the labeling of hepatic glycogen pools that subsequently could be depleted (with concomitant ({sup 14}C)glucose release) by either glucagon or norepinephrine. Aftermore » the addition of a highly concentrated human monocyte-conditioned medium (MCM) or various cytokines to these prelabeled cells, ({sup 14}C)glucose release was stimulated by MCM and recombinant human interleukin 6 (IL-6) but was not stimulated by other cytokines tested. Furthermore, only antisera to IL-6 were capable of reducing the glucose-releasing factor activity found in MCM. These data therefore suggest a novel glucoregulatory role for IL-6.« less

Irreversible inhibition of BTK kinase by a novel highly selective inhibitor CHMFL-BTK-11 suppresses inflammatory response in rheumatoid arthritis model.

PubMed

Wu, Hong; Huang, Qiong; Qi, Ziping; Chen, Yongfei; Wang, Aoli; Chen, Cheng; Liang, Qianmao; Wang, Jinghua; Chen, Wensheng; Dong, Jin; Yu, Kailin; Hu, Chen; Wang, Wenchao; Liu, Xiaochuan; Deng, Yuanxin; Wang, Li; Wang, Beilei; Li, Xiaoxiang; Gray, Nathanael S; Liu, Jing; Wei, Wei; Liu, Qingsong

2017-03-28

BTK plays a critical role in the B cell receptor mediated inflammatory signaling in the rheumatoid arthritis (RA). Through a rational design approach we discovered a highly selective and potent BTK kinase inhibitor (CHMFL-BTK-11) which exerted its inhibitory efficacy through a covalent bond with BTK Cys481. CHMFL-BTK-11 potently blocked the anti-IgM stimulated BCR signaling in the Ramos cell lines and isolated human primary B cells. It significantly inhibited the LPS stimulated TNF-α production in the human PBMC cells but only weakly affecting the normal PBMC cell proliferation. In the adjuvant-induced arthritis rat model, CHMFL-BTK-11 ameliorated the inflammatory response through blockage of proliferation of activated B cells, inhibition of the secretion of the inflammatory factors such as IgG1, IgG2, IgM, IL-6 and PMΦ phagocytosis, stimulation of secretion of IL-10. The high specificity of CHMFL-BTK-11 makes it a useful pharmacological tool to further detect BTK mediated signaling in the pathology of RA.

Activation of platelet-rich plasma using soluble type I collagen.

PubMed

Fufa, Duretti; Shealy, Blake; Jacobson, May; Kevy, Sherwin; Murray, Martha M

2008-04-01

Platelet-rich plasma (PRP) has recently been found to be a useful delivery system for growth factors important to oral tissue healing. But application of PRP in a liquid form to a wound site within the oral cavity can be complicated by significant loss of the PRP into the surrounding oral space unless gelation through the clotting mechanism is accomplished. Gelation is currently accomplished using bovine thrombin; however, rare but serious complications of this method have led to the search for alternative clotting mechanisms, including the use of soluble collagen as a clotting activator. In this work, our hypothesis was that soluble type I collagen would be as effective as bovine thrombin in causing clotting of the PRP and stimulating growth factor release from the platelets and granulocytes. PRP from human donors was clotted using type I collagen or bovine thrombin. Clot retraction was determined by measuring clot diameters over time. The release of platelet-derived growth factor (PDGF)-AB, transforming growth factor (TGF)-beta1, and vascular endothelial growth factor (VEGF) from both types of clots was measured over 10 days using enzyme-linked immunosorbent assasy. Clots formed using type I collagen exhibited far less retraction than those formed with bovine thrombin. Bovine thrombin and type I collagen stimulated similar release of PDGF-AB and VEGF between 1 and 10 days; however, thrombin activation resulted in a greater release of TGF-beta1 during the first 5 days after activation. The use of type I collagen to activate clotting of PRP may be a safe and effective alternative to bovine thrombin. The use of collagen results in less clot retraction and equal release of PDGF-AB and VEGF compared with currently available methods of clot activation.

Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C and mitogenic factor MF.

PubMed Central

Schmidt, K H; Gerlach, D; Wollweber, L; Reichardt, W; Mann, K; Ozegowski, J H; Fleischer, B

1995-01-01

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein. PMID:7591107

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin–vascular endothelial growth factor pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhishan; Humphries, Brock; Xiao, Hua

2013-08-15

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchialmore » epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro-angiogenic effect. • EMT in arsenic-transformed cells activates β-catenin. • β-Catenin activation increases VEGF expression in arsenic-transformed cells. • β-Catenin activation is likely independent of canonical Wnt signaling. • EMT in arsenic-transformed cells promotes angiogenesis via β-catenin–VEGF pathway.« less

Krüppel-like factor 4 regulates the expression of inducible nitric oxide synthase induced by TNF-α in human fibroblast-like synoviocyte MH7A cells.

PubMed

Mo, Xuanrong; Chen, Jie; Wang, Xinjuan; Pan, Zhenyu; Ke, Yuping; Zhou, Zhidong; Xie, Jiangwen; Lv, Guoju; Luo, Xinjing

2018-01-01

Krüppel-like factor 4 (KLF4), a zinc finger transcription factor, has been implicated in the inflammation mediated by macrophages and endothelial cells by regulating the expression of inflammatory mediators. Here, we investigated whether KLF4 affects the expression of inducible nitric oxide synthase (iNOS), an important inflammatory mediator, in the human RA fibroblast-like synovial cell line MH7A. A pcDNA3.1-KLF4 plasmid or short interfering RNA KLF4 was transfected into MH7A cells, and the iNOS expression and nitric oxide (NO) production were analyzed by quantitative PCR, immunoblotting, and nitrite measurement. The iNOS promoter activity was determined by luciferase assay. The results showed overexpression of KLF4 increased iNOS expression and NO production in the presence or absence of TNF-α. Conversely, KLF4 knockdown markedly reduced iNOS expression and NO production induced by TNF-α. KLF4 activated the transcription activity of iNOS promoter in MH7A cells stimulated by TNF-α. This study indicates that KLF4 is important for regulating the expression of iNOS by TNF-α in human synoviocytes.

Cyclic AMP-dependent modification of gonad-selective TAF(II)105 in a human ovarian granulosa cell line.

PubMed

Wu, Yimin; Lu, Yunzhe; Hu, Yanfen; Li, Rong

2005-11-01

In response to gonadotropins, the elevated level of intracellular-cyclic AMP (cAMP) in ovarian granulosa cells triggers an ordered activation of multiple ovarian genes, which in turn promotes various ovarian functions including folliculogenesis and steroidogenesis. Identification and characterization of transcription factors that control ovarian gene expression are pivotal to the understanding of the molecular basis of the tissue-specific gene regulation programs. The recent discovery of the mouse TATA binding protein (TBP)-associated factor 105 (TAF(II)105) as a gonad-selective transcriptional co-activator strongly suggests that general transcription factors such as TFIID may play a key role in regulating tissue-specific gene expression. Here we show that the human TAF(II)105 protein is preferentially expressed in ovarian granulosa cells. We also identified a novel TAF(II)105 mRNA isoform that results from alternative exon inclusion and is predicted to encode a dominant negative mutant of TAF(II)105. Following stimulation by the adenylyl cyclase activator forskolin, TAF(II)105 in granulosa cells undergoes rapid and transient phosphorylation that is dependent upon protein kinase A (PKA). Thus, our work suggests that pre-mRNA processing and post-translational modification represent two important regulatory steps for the gonad-specific functions of human TAF(II)105. Copyright 2005 Wiley-Liss, Inc.

PROPERTIES OF VARIOUS ANTI-γ-GLOBULIN FACTORS IN HUMAN SERA

PubMed Central

Harboe, Morten; Rau, Barbara; Aho, Kimmo

1965-01-01

The serological and physicochemical properties of the following three forms of human anti-γ-globulin factors were compared: (a) rheumatoid factors; (b) Milgrom type anti-γ-globulin factors; and (c) factors directed against an antigen in human γG-globulin that is hidden in the intact molecule and revealed by enzymatic digestion at low pH. The property common to these factors is ability to interact with human γG-globulin; they are distinguishable because they react with different antigenic groups on this molecule. In all of five sera, the Milgrom type anti-γ-globulin factors were γM-globulins. They reacted with various human γG-globulin antibodies but failed to interact with γM-globulin type antibodies in agglutination and absorption experiments. When isolated from other anti-γ-globulin factors, they agglutinated red cells coated with intact anti-Rh antibodies, but failed to react with cells cells coated with pepsin-digested anti-Rh antibody. These observations indicate that the agglutinator reacts with the crystallizable, inert fragment of γG-globulin. Anti-γ-globulin activity directed against an antigen in human γG-globulin revealed by pepsin digestion was demonstrated in γG-, γA-, and γM-globulins. This anti-γ-globulin factor could be absorbed by antigen-antibody precipitates containing human antibody, which shows that the hidden antigen in human γG-globulin is revealed not only by enzymatic digestion at low pH, but also when γG-globulin is present as antibody in an antigen-antibody precipitate. Rheumatoid factors and Milgrom type anti-γ-globulin factors were also absorbed by antigen-antibody precipitates containing human antibody. The results indicate that the three distinct forms of antiγ-globulin factors may all be produced as a result of antigenic stimulation by autologous antigen-antibody complexes. PMID:14276773

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases

PubMed Central

Lesslie, D P; Summy, J M; Parikh, N U; Fan, F; Trevino, J G; Sawyer, T K; Metcalf, C A; Shakespeare, W C; Hicklin, D J; Ellis, L M; Gallick, G E

2006-01-01

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process. PMID:16685275

Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc gamma receptor-specific mechanisms.

PubMed

Crockett-Torabi, E; Fantone, J C

1990-11-01

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor.

N-acetylcysteine attenuates TNF-α-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

PubMed Central

Hashimoto, Shu; Gon, Yasuhiro; Matsumoto, Ken; Takeshita, Ikuko; Horie, Takashi

2001-01-01

We have previously shown that tumour necrosis factor-α (TNF-α) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF-α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-α-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC-treated cells. NAC attenuated TNF-α-induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-α-stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury. PMID:11156586

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

PubMed

Hashimoto, S; Gon, Y; Matsumoto, K; Takeshita, I; Horie, T

2001-01-01

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Characterization of synthetic lung surfactant activity against proinflammatory cytokines in human monocytes.

PubMed

Otsubo, Eiji; Irimajiri, Kiyohiro; Takei, Tsunetomo; Nomura, Masato

2002-03-01

Our previous study demonstrated that the smallest synthetic peptide with the sequence CPVHLKRLLLLLLLLLLLLLLLL, SP-CL16(6-28), admixed with phospholipid (synthetic lung surfactant, SLS) showed strong surface activity. In this study, we attempted to develop a dual-type surfactant with both anti inflammatory and surface activities. SP-CL16(6-28) was first chemically synthesized and then purified for use by centrifugal partition chromatography. A mixture of SP-CL16(6-28) and phospholipid complex was tested for anti inflammatory activity using the human monocyte cell line THP-1. Whether the suppression of tumor necrosis factor-alpha (TNF-a), interleukin (IL)-8, IL-6, IL-1beta, and macrophage migration inhibitory factor (MIF) was reduced by lipopolysaccharide (LPS) in monocytes was examined. Levels of these cytokines were measured by enzyme-linked immunosorbent assay. It was found that SLS significantly and dose dependently inhibited the secretion of TNF-alpha by THP-1 cells following stimulation with LPS. Dipalmitoylphosphatidylcoline did not inhibit the release of cytokines. These findings suggest that SLS has anti inflammatory activity. Therefore it should be possible to develop a SLS with both anti inflammatory activity and surface activity.

Plasma rich in growth factors (PRGF-Endoret) reduces neuropathologic hallmarks and improves cognitive functions in an Alzheimer's disease mouse model.

PubMed

Anitua, Eduardo; Pascual, Consuelo; Antequera, Desiree; Bolos, Marta; Padilla, Sabino; Orive, Gorka; Carro, Eva

2014-07-01

Impaired growth factor function is thought to drive many of the alterations observed in Alzheimer's disease (AD) patients. Endogenous regenerative technology, PRGF (plasma rich in growth factor)-Endoret, is designed for the delivery of a complex pool of patient's own active morphogens that may stimulate tissue regeneration. We obtained and characterized PRGF-Endoret preparations from human blood. We used, as experimental approach in vivo, APP/PS1 mice, characterized by age-dependent brain amyloid-β (Aβ) accumulation. Intranasal administration of PRGF-Endoret to APP/PS1 mice resulted in an important decrease in brain Aβ deposition and tau phosphorylation. PRGF-Endoret-treated APP/PS1 mice also showed decreased astrocyte reactivity, and prevented protein synaptic loss. In vitro approaches demonstrated that PRGF-Endoret treatment modulated astrocyte activation, reducing inflammatory responses, and promoted Aβ degradation. Furthermore, PRGF-Endoret stimulated global improvements in anxiety, learning, and memory behaviors. Our findings show that PRGF-Endoret exerts multifunctional and complementary effects that result in the reversal of the broad range of cognitive deficits in AD, suggesting that PRGF-Endoret may hold promise as an innovative therapy in AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus

PubMed Central

Markus, Steven M.; Taneja, Samir S.; Logan, Susan K.; Li, Wenhui; Ha, Susan; Hittelman, Adam B.; Rogatsky, Inez; Garabedian, Michael J.

2002-01-01

The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR153–336, containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR153–336 fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation. PMID:11854421

VPS34 stimulation of p62 phosphorylation for cancer progression.

PubMed

Jiang, X; Bao, Y; Liu, H; Kou, X; Zhang, Z; Sun, F; Qian, Z; Lin, Z; Li, X; Liu, X; Jiang, L; Yang, Y

2017-12-14

Vps34, a class III PtdIns3 lipid kinase involved in the control of both autophagic and endocytic systems, has been studied extensively in numerous fundamental cellular processes. Accumulating evidence indicates that Vps34 may also contribute to the development and progression of human cancers. However, the mechanism of Vps34 in tumorigenesis remains elusive. Here, we report an unanticipated role of Vps34 in the activation of p62 for cancer development. We identified that Vps34 is a transcriptional activator of p62 through competition of Nrf2 (nuclear factor erythroid 2-related factor 2) for Keap1 binding. Vps34 augments the association of PKC-δ with p62 for its phosphorylation at Serine 349, which leads to positive feedback on the Nrf2-dependent transcription of oncogenes. Additionally, we found that the expression of Vps34 is correlated with the tumorigenic activity of human breast cancer cells. Normally inactive in breast cancer, caspase 8 can cleave Vps34 at residue D285, which directly abolished its lipid kinase activity and dramatically altered cell invasion potential, colony formation, as well as tumorigenesis in orthotopic engraftments in mice. The cleavage at D285 blocks expression of LC3-II, Nrf2 and subsequently, p62, in addition to blocking tumor growth, indicating that the intact structure of Vps34 is essential for its activity. Moreover, either knockout of PKC-δ or knockdown of p62 by small interfering RNA in MCF-7 cells abrogates Vps34-dependent tumor growth. Data presented here suggested that Vps34 stimulates tumor development mainly through PKC-δ- activation of p62.

Dual agonist Surrobody™ simultaneously activates death receptors DR4 and DR5 to induce cancer cell death

PubMed Central

Milutinovic, Snezana; Kashyap, Arun K.; Yanagi, Teruki; Wimer, Carina; Zhou, Sihong; O' Neil, Ryann; Kurtzman, Aaron L.; Faynboym, Alexsandr; Xu, Li; Hannum, Charles H.; Diaz, Paul W.; Matsuzawa, Shu-ichi; Horowitz, Michael; Horowitz, Lawrence; Bhatt, Ramesh R.; Reed, John C.

2015-01-01

Death receptors of the Tumor Necrosis Factor (TNF) family are found on surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors-4 and -5 (DR4 and DR5) is Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, TRAIL (Apo2L). Since most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody™ technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing pro-apoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 mono-specific antibodies. Taken together, Surrobody shows promising preclinical pro-apoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent. PMID:26516157

Identification and characterization of ART-27, a novel coactivator for the androgen receptor N terminus.

PubMed

Markus, Steven M; Taneja, Samir S; Logan, Susan K; Li, Wenhui; Ha, Susan; Hittelman, Adam B; Rogatsky, Inez; Garabedian, Michael J

2002-02-01

The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR(153-336), containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR(153-336) fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.

HIV-1 evades innate immune recognition through specific cofactor recruitment

NASA Astrophysics Data System (ADS)

Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.

2013-11-01

Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

The control of vascular endothelial cell injury.

PubMed

Murota, S; Morita, I; Suda, N

1990-01-01

The mechanism by which MCI-186 showed a potent cytoprotective effect on the in vitro endothelial cell injury due to 15-HPETE was studied. Stimulation of human leukocytes with various chemical mediators such as TPA, f-Met-Leu-Phe, LTB4, etc. elicited the production of active oxygens, which could be detected by luminol-dependent chemiluminescence. Among the chemical mediators tested, TPA elicited the chemiluminescence the most, and f-Met-Leu-Phe and LTB4 came next. When the leukocytes were directly placed on a monolayer of cultured endothelial cells, followed by stimulating the leukocytes with TPA, severe endothelial cell injury was observed. The effect of TPA was dose dependent. There was good correlation between the active oxygen releasing activity and the cytotoxic activity. When the leukocytes were placed on a filter which was set apart from the monolayer of endothelial cell in a culture dish, and stimulated the leukocytes with TPA, no cytotoxicity was observed. These data strongly suggest that the substance responsible for the cytotoxicity must be a very labile and short-lived substance, presumably active oxygens. On the other hand, MCI-186 was found to have a complete quenching activity to the chemiluminescence due to active oxygens in the TPA-leukocyte system. Taken together, these factors indicate that the potent cytoprotective effect of MCI-186 may be due to its specific radical scavenging activity.

Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells.

PubMed

Márquez, Saioa; Fernández, José Javier; Terán-Cabanillas, Eli; Herrero, Carmen; Alonso, Sara; Azogil, Alicia; Montero, Olimpio; Iwawaki, Takao; Cubillos-Ruiz, Juan R; Fernández, Nieves; Crespo, Mariano Sánchez

2017-01-01

Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans -activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs.

Endoplasmic Reticulum Stress Sensor IRE1α Enhances IL-23 Expression by Human Dendritic Cells

PubMed Central

Márquez, Saioa; Fernández, José Javier; Terán-Cabanillas, Eli; Herrero, Carmen; Alonso, Sara; Azogil, Alicia; Montero, Olimpio; Iwawaki, Takao; Cubillos-Ruiz, Juan R.; Fernández, Nieves; Crespo, Mariano Sánchez

2017-01-01

Human monocyte-derived dendritic cells (DCs) exposed to pathogen-associated molecular patterns (PAMPs) undergo bioenergetic changes that influence the immune response. We found that stimulation with PAMPs enhanced glycolysis in DCs, whereas oxidative phosphorylation remained unaltered. Glucose starvation and the hexokinase inhibitor 2-deoxy-d-glucose (2-DG) modulated cytokine expression in stimulated DCs. Strikingly, IL23A was markedly induced upon 2-DG treatment, but not during glucose deprivation. Since 2-DG can also rapidly inhibit protein N-glycosylation, we postulated that this compound could induce IL-23 in DCs via activation of the endoplasmic reticulum (ER) stress response. Indeed, stimulation of DCs with PAMPs in the presence of 2-DG robustly activated inositol-requiring protein 1α (IRE1α) signaling and to a lesser extent the PERK arm of the unfolded protein response. Additional ER stressors such as tunicamycin and thapsigargin also promoted IL-23 expression by PAMP-stimulated DCs. Pharmacological, biochemical, and genetic analyses using conditional knockout mice revealed that IL-23 induction in ER stressed DCs stimulated with PAMPs was IRE1α/X-box binding protein 1-dependent upon zymosan stimulation. Interestingly, we further evidenced PERK-mediated and CAAT/enhancer-binding protein β-dependent trans-activation of IL23A upon lipopolysaccharide treatment. Our findings uncover that the ER stress response can potently modulate cytokine expression in PAMP-stimulated human DCs. PMID:28674530

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

PubMed Central

Adams, A F; Haslam, R J

1978-01-01

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors. PMID:29607

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

PubMed

Adams, A F; Haslam, R J

1978-07-15

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.

Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport.

PubMed

Fischer, C P; Bode, B P; Takahashi, K; Tanabe, K K; Souba, W W

1996-05-01

The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL-6 receptor expression on the cell surface. It is likely that this upregulation of IL-6 receptors "primes" human liver cells for subsequent stimulation by cytokines. The resulting increase in hepatic amino acid transport provides the liver with substrate to support key metabolic pathways during catabolic states.

Transcranial magnetic and electrical stimulation compared: does TES activate intracortical neuronal circuits?

PubMed

Brocke, J; Irlbacher, K; Hauptmann, B; Voss, M; Brandt, S A

2005-12-01

To determine whether, and under which conditions, transcranial electrical stimulation (TES) and transcranial magnetic stimulation (TMS) can activate similar neuronal structures of the human motor cortex, as indicated by electromyographic recordings. Focal TMS was performed on three subjects inducing a postero-anterior directed current (p-a), TES with postero-anteriorly (p-a) and latero-medially (l-m) oriented electrodes. We analyzed the onset latencies and amplitudes (single-pulse) and intracortical inhibition and excitation (paired-pulse). TMS p-a and TES p-a produced muscle responses with the same onset latency, while TES l-m led to 1.4-1.9 ms shorter latencies. Paired-pulse TMS p-a and TES p-a induced inhibition at short inter-stimulus intervals (ISI) (maximum: 2-3 ms) and facilitation at longer ISIs (maximum: 10 ms). No inhibition but a strong facilitation was obtained from paired-pulse TES l-m (ISIs 1-5 ms). Our findings support the hypothesis, that current direction is the most relevant factor in determining the mode of activation for both TMS and TES: TMS p-a and TES p-a are likely to activate the corticospinal neurons indirectly. In contrast, TES l-m may preferentially activate the corticospinal fibres directly, distant of the neuronal body. TES is a suitable tool to induce intracortical inhibition and excitation.

Whitening and anti-wrinkle activities of ferulic acid isolated from Tetragonia tetragonioides in B16F10 melanoma and CCD-986sk fibroblast cells.

PubMed

Park, Hye-Jin; Cho, Jun-Hyo; Hong, Shin-Hyub; Kim, Dong-Hee; Jung, Hee-Young; Kang, In-Kyu; Cho, Young-Je

2018-01-01

Ferulic acid isolated from Tetragonia tetragonioides was tested for its whitening effect on the B16F10 mouse melanoma cell line and its anti-wrinkle activity on the CCD-986sk human dermal fibroblast cell line. Ferulic acid, one of the primary phenolic compounds that can be isolated from T. tetragonioides, has been reported to show potential as a functional food, for its whitening effect and anti-wrinkle activity. To measure its whitening and anti-wrinkle activities, cells were treated with ferulic acid isolated from T. tetragonioides at concentrations between 5 and 20 μM. Ferulic acid showed no cytotoxicity at concentrations up to 20 μM. Ferulic acid inhibited melanin synthesis, tyrosinase expression, and microphthalmia transcription factor expression in B16F10 cells stimulated with α-melanocyte stimulating hormone. Ferulic acid induced procollagen synthesis, hyaluronic acid synthesis, tissue inhibitor of metalloproteinase synthesis, and inhibited matrix metalloproteinase (MMP)-1 and MMP-9 expression in CCD-986sk cells stimulated with UV-B. On the basis of these results, we conclude that ferulic acid isolated from T. tetragonioides shows potential for use as a functional food, with whitening and anti-wrinkle activities.

Structural basis for the recognition of Asef by adenomatous polyposis coli

PubMed Central

Zhang, Zhenyi; Chen, Leyi; Gao, Lei; Lin, Kui; Zhu, Liang; Lu, Yang; Shi, Xiaoshan; Gao, Yuan; Zhou, Jing; Xu, Ping; Zhang, Jian; Wu, Geng

2012-01-01

Adenomatous polyposis coli (APC) regulates cell-cell adhesion and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (GEF; Asef), which is usually autoinhibited through the binding between its Src homology 3 (SH3) and Dbl homology (DH) domains. The APC-activated Asef stimulates the small GTPase Cdc42, which leads to decreased cell-cell adherence and enhanced cell migration. In colorectal cancers, truncated APC constitutively activates Asef and promotes cancer cell migration and angiogenesis. Here, we report crystal structures of the human APC/Asef complex. We find that the armadillo repeat domain of APC uses a highly conserved surface groove to recognize the APC-binding region (ABR) of Asef, conformation of which changes dramatically upon binding to APC. Key residues on APC and Asef for the complex formation were mutated and their importance was demonstrated by binding and activity assays. Structural superimposition of the APC/Asef complex with autoinhibited Asef suggests that the binding between APC and Asef might create a steric clash between Asef-DH domain and APC, which possibly leads to a conformational change in Asef that stimulates its GEF activity. Our structures thus elucidate the molecular mechanism of Asef recognition by APC, as well as provide a potential target for pharmaceutical intervention against cancers. PMID:21788986

Simultaneous transcranial magnetic stimulation and single neuron recording in alert non-human primates

PubMed Central

Mueller, Jerel K.; Grigsby, Erinn M.; Prevosto, Vincent; Petraglia, Frank W.; Rao, Hrishikesh; Deng, Zhi-De; Peterchev, Angel V.; Sommer, Marc A.; Egner, Tobias; Platt, Michael L.; Grill, Warren M.

2014-01-01

Transcranial magnetic stimulation (TMS) is a widely used, noninvasive method for stimulating nervous tissue, yet its mechanisms of effect are poorly understood. Here we report novel methods for studying the influence of TMS on single neurons in the brain of alert non-human primates. We designed a TMS coil that focuses its effect near the tip of a recording electrode and recording electronics that enable direct acquisition of neuronal signals at the site of peak stimulus strength minimally perturbed by stimulation artifact in intact, awake monkeys (Macaca mulatta). We recorded action potentials within ~1 ms after 0.4 ms TMS pulses and observed changes in activity that differed significantly for active stimulation as compared to sham stimulation. The methodology is compatible with standard equipment in primate laboratories, allowing for easy implementation. Application of these new tools will facilitate the refinement of next generation TMS devices, experiments, and treatment protocols. PMID:24974797

Role of granulocyte colony-stimulating factor in human reproduction.

PubMed

Eftekhar, Maryam; Naghshineh, Elham; Khani, Parisa

2018-01-01

As new research reveals, granulocyte colony-stimulating factor (G-CSF) plays an effective role in pregnancy success, considering that it not only affects the embryo implantation and ovarian function but also it promotes endometrial thickening and improves the pathophysiology of endometriosis, which all fundamentally lead to reducing pregnancy loss. In this review, we focus on the role of G-CSF in human reproduction. We summarized its role in ovulation, luteinized unruptured follicle syndrome, poor responders, improving repeated in vitro fertilization failure, endometrial receptivity and treatment of thin endometrium, and recurrent spontaneous abortion.

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

PubMed Central

Guessous, Fadila; Marcinkiewicz, Marek; Polanowska-Grabowska, Renata; Kongkhum, Sudawadee; Heatherly, Daniel; Obrig, Tom; Gear, Adrian R. L.

2005-01-01

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1α (SDF-1α) or SDF-1β and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1α at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1α, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS. PMID:16299328

In vitro evaluation of the synergistic antioxidant and anti-inflammatory activities of the combined extracts from Malaysian Ganoderma lucidum and Egyptian Chlorella vulgaris.

PubMed

Abu-Serie, Marwa M; Habashy, Noha H; Attia, Wafaa E

2018-05-10

Since oxidative stress and inflammation are two linked factors in the pathogenesis of several human diseases. Thus identification of effective treatment is of great importance. Edible mushroom and microalgae are rich in the effective antioxidant phytochemicals. Hence, their beneficial effects on oxidative stress-associated inflammation are extremely required to be investigated. This study evaluated the functional constituents, antioxidant and anti-inflammatory activities of Malaysian Ganoderma lucidum aqueous extract (GLE) and Egyptian Chlorella vulgaris ethanolic extract (CVE). Also, the synergistic, addictive or antagonistic activities of the combination between the two extracts (GLE-CVE) were studied. Expression of inducible nitric oxide synthase, cyclooxygenase-2, and nuclear factor-kappa B, as well as levels of nitric oxide, tumor necrosis factor (TNF)-α, lipid peroxidation, reduced glutathione and antioxidant enzymes were determined using in vitro model of lipopolysaccharide-stimulated white blood cells.

RANTES release by human airway smooth muscle: effects of prostaglandin E(2) and fenoterol.

PubMed

Lazzeri, N; Belvisi, M G; Patel, H J; Chung, K F; Yacoub, M H; Mitchell, J A

2001-12-21

In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

PubMed Central

Hernández, Damián; Millard, Rodney; Sivakumaran, Priyadharshini; Wong, Raymond C. B.; Crombie, Duncan E.; Hewitt, Alex W.; Liang, Helena; Hung, Sandy S. C.; Pébay, Alice; Shepherd, Robert K.; Dusting, Gregory J.; Lim, Shiang Y.

2016-01-01

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used. PMID:26788064

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Marquez, Maribel P; Alencastro, Frances; Madrigal, Alma; Jimenez, Jossue Loya; Blanco, Giselle; Gureghian, Alex; Keagy, Laura; Lee, Cecilia; Liu, Robert; Tan, Lun; Deignan, Kristen; Armstrong, Brian; Zhao, Yuanxiang

2017-11-01

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Interleukin-1β inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts.

PubMed

Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

2013-12-05

Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. Published by Elsevier Ireland Ltd.

Interleukin-1β Inhibits Insulin Signaling and Prevents Insulin-Stimulated System A Amino Acid Transport in Primary Human Trophoblasts

PubMed Central

Aye, Irving L. M. H.; Jansson, Thomas; Powell, Theresa L.

2013-01-01

Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10 pg/ml) for 24 hours. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. PMID:23891856

Safety, pharmacokinetics, and pharmacodynamics of RSLV-132, an RNase-Fc fusion protein in systemic lupus erythematosus: a randomized, double-blind, placebo-controlled study.

PubMed

Burge, D J; Eisenman, J; Byrnes-Blake, K; Smolak, P; Lau, K; Cohen, S B; Kivitz, A J; Levin, R; Martin, R W; Sherrer, Y; Posada, J A

2017-07-01

Blood-borne RNA circulating in association with autoantibodies is a potent stimulator of interferon production and immune system activation. RSLV-132 is a novel fully human biologic Fc fusion protein that is comprised of human RNase fused to the Fc domain of human IgG1. The drug is designed to remain in circulation and digest extracellular RNA with the aim of preventing activation of the immune system via Toll-like receptors and the interferon pathway. The present study describes the first clinical study of nuclease therapy in 32 subjects with systemic lupus erythematosus. The drug was well tolerated with a very favorable safety profile. The approximately 19-day serum half-life potentially supports once monthly dosing. There were no subjects in the study that developed anti-RSLV-132 antibodies. Decreases in B-cell activating factor correlated with decreases in disease activity in a subset of patients.

Gemfibrozil, a lipid lowering drug, inhibits the activation of primary human microglia via peroxisome proliferator-activated receptor β.

PubMed

Jana, Malabendu; Pahan, Kalipada

2012-08-01

Microglial activation participates in the pathogenesis of various neuroinflammatory and neurodegenerative diseases. However, mechanisms by which microglial activation could be controlled are poorly understood. Peroxisome proliferator-activated receptors (PPAR) are transcription factors belonging to the nuclear receptor super family with diverse effect. This study underlines the importance of PPARβ/δ in mediating the anti-inflammatory effect of gemfibrozil, an FDA-approved lipid-lowering drug, in primary human microglia. Bacterial lipopolysachharides (LPS) induced the expression of various proinflammatory molecules and upregulated the expression of microglial surface marker CD11b in human microglia. However, gemfibrozil markedly suppressed proinflammatory molecules and CD11b in LPS-stimulated microglia. Human microglia expressed PPAR-β and -γ, but not PPAR-α. Interestingly, either antisense knockdown of PPAR-β or antagonism of PPAR-β by a specific chemical antagonist abrogated gemfibrozil-mediated inhibition of microglial activation. On the other hand, blocking of PPAR-α and -γ had no effect on gemfibrozil-mediated anti-inflammatory effect in microglia. These results highlight the fact that gemfibrozil regulates microglial activation by inhibiting inflammatory gene expression in a PPAR-β dependent pathway and further reinforce its therapeutic application in several neuroinflammatory and neurodegenerative diseases.

Reg proteins and their roles in inflammation and cancer of the human digestive system.

PubMed

Zhao, Jie; Wang, Jingyu; Wang, Hao; Lai, Maode

2013-01-01

The regenerating gene (Reg) family is a group of small molecules that includes four members found in various species, although only three are found in human tissues. Their expression is stimulated by certain growth factors or cytokines. The Reg family plays different roles in proliferation, migration, and anti-apoptosis through activating different signaling pathways. Their dysexpression is closely associated with a number of human conditions and diseases such as inflammation and cancer, especially in the human digestive system. Clinically, upregulation of Reg proteins is usually demonstrated in histological sections and sera from cancer patients. Therefore, Reg proteins can predict the progression and prognosis of cancers, especially those of the digestive tract, and can also act as diagnostic markers and therapeutic targets.

Homeobox B9 is overexpressed in hepatocellular carcinomas and promotes tumor cell proliferation both in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Fangyi; Dong, Lei, E-mail: dlleidong@126.com; Xing, Rong

2014-02-07

Highlights: • HOXB9 is overexpressed in human HCC samples. • HOXB9 over expression had shorter survival time than down expression. • HOXB9 stimulated the proliferation of HCC cells. • Activation of TGF-β1 contributes to HOXB9-induced proliferation in HCC cells. - Abstract: HomeoboxB9 (HOXB9), a nontransforming transcription factor that is overexpressed in multiple tumor types, alters tumor cell fate and promotes tumor progression. However, the role of HOXB9 in hepatocellular carcinoma (HCC) development has not been well studied. In this paper, we found that HOXB9 is overexpressed in human HCC samples. We investigated HOXB9 expression and its prognostic value for HCC.more » HCC surgical tissue samples were taken from 89 HCC patients. HOXB9 overexpression was observed in 65.2% of the cases, and the survival analysis showed that the HOXB9 overexpression group had significantly shorter overall survival time than the HOXB9 downexpression group. The ectopic expression of HOXB9 stimulated the proliferation of HCC cells; whereas the knockdown of HOXB9 produced an opposite effect. HOXB9 also modulated the tumorigenicity of HCC cells in vivo. Moreover, we found that the activation of TGF-β1 contributes to HOXB9-induced proliferation activities. The results provide the first evidence that HOXB9 is a critical regulator of tumor growth factor in HCC.« less

Possible mechanism for preterm labor associated with bacterial infection. I. Stimulation of phosphoinositide metabolism by endotoxin in endometrial fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, A.A.; Imai, A.; Tamaya, T.

Growing evidence suggests an association between intra-amniotic infection and premature initiation of parturition. We recently demonstrated that some factor(s) including endotoxin produced by the organism stimulates endogenous phospholipase A2 resulting in liberation of arachidonic acid and prostaglandin formation. The studies presented in this report were designated to evaluate the mechanism for endotoxin to stimulate phospholipase A2 using human endometrial fibroblasts. Exposure of the fibroblasts to endotoxin from Escherichia coli in the presence of ({sup 32}P) phosphate increased {sup 32}P-labeling of phosphatidic acid (PA) and phosphatidyl-inositol (PI) in a dose-dependent and a time-dependent manners. The PA labeling occurred without a measurablemore » lag time. These findings demonstrate that the endotoxin stimulates phosphoinositide metabolism in human endometrial fibroblasts by a receptor-mediated mechanism. Membrane phosphoinositide turnover stimulated by endotoxin results in cytosolic Ca{sup 2+} increment, liberation of arachidonic acid, which may be involved in the initiation of parturition.« less

Tissue engineering skeletal muscle for orthopaedic applications

NASA Technical Reports Server (NTRS)

Payumo, Francis C.; Kim, Hyun D.; Sherling, Michael A.; Smith, Lee P.; Powell, Courtney; Wang, Xiao; Keeping, Hugh S.; Valentini, Robert F.; Vandenburgh, Herman H.

2002-01-01

With current technology, tissue-engineered skeletal muscle analogues (bioartificial muscles) generate too little active force to be clinically useful in orthopaedic applications. They have been engineered genetically with numerous transgenes (growth hormone, insulinlike growth factor-1, erythropoietin, vascular endothelial growth factor), and have been shown to deliver these therapeutic proteins either locally or systemically for months in vivo. Bone morphogenetic proteins belonging to the transforming growth factor-beta superfamily are osteoinductive molecules that drive the differentiation pathway of mesenchymal cells toward the chondroblastic or osteoblastic lineage, and stimulate bone formation in vivo. To determine whether skeletal muscle cells endogenously expressing bone morphogenetic proteins might serve as a vehicle for systemic bone morphogenetic protein delivery in vivo, proliferating skeletal myoblasts (C2C12) were transduced with a replication defective retrovirus containing the gene for recombinant human bone morphogenetic protein-6 (C2BMP-6). The C2BMP-6 cells constitutively expressed recombinant human bone morphogenetic protein-6 and synthesized bioactive recombinant human bone morphogenetic protein-6, based on increased alkaline phosphatase activity in coincubated mesenchymal cells. C2BMP-6 cells did not secrete soluble, bioactive recombinant human bone morphogenetic protein-6, but retained the bioactivity in the cell layer. Therefore, genetically-engineered skeletal muscle cells might serve as a platform for long-term delivery of osteoinductive bone morphogenetic proteins locally.

Age-Related Gene Expression Differences in Monocytes from Human Neonates, Young Adults, and Older Adults

PubMed Central

Tong, Ann-Jay; Kollmann, Tobias R.; Smale, Stephen T.

2015-01-01

A variety of age-related differences in the innate and adaptive immune systems have been proposed to contribute to the increased susceptibility to infection of human neonates and older adults. The emergence of RNA sequencing (RNA-seq) provides an opportunity to obtain an unbiased, comprehensive, and quantitative view of gene expression differences in defined cell types from different age groups. An examination of ex vivo human monocyte responses to lipopolysaccharide stimulation or Listeria monocytogenes infection by RNA-seq revealed extensive similarities between neonates, young adults, and older adults, with an unexpectedly small number of genes exhibiting statistically significant age-dependent differences. By examining the differentially induced genes in the context of transcription factor binding motifs and RNA-seq data sets from mutant mouse strains, a previously described deficiency in interferon response factor-3 activity could be implicated in most of the differences between newborns and young adults. Contrary to these observations, older adults exhibited elevated expression of inflammatory genes at baseline, yet the responses following stimulation correlated more closely with those observed in younger adults. Notably, major differences in the expression of constitutively expressed genes were not observed, suggesting that the age-related differences are driven by environmental influences rather than cell-autonomous differences in monocyte development. PMID:26147648

IL-33 and IgE stimulate mast cell production of IL-2 and regulatory T cell expansion in allergic dermatitis.

PubMed

Salamon, P; Shefler, I; Moshkovits, I; Munitz, A; Horwitz Klotzman, D; Mekori, Y A; Hershko, A Y

2017-11-01

We have previously shown that mast cells (MCs) suppress chronic allergic dermatitis in mice. The underlying mechanism involves MC-derived IL-2, which supports regulatory T cell (Treg) response at the site of inflammation. However, it is not clear what are the factors that drive MCs to produce IL-2. To understand the mechanisms that lead to IL-2 production from MCs in chronic allergic dermatitis. Isolated murine bone marrow-derived MCs (BMMCs) were incubated with various stimulators, and IL-2 production was assessed by RT-PCR and ELISA. The response of signalling pathways was evaluated by MAPK inhibitors and Western blot analysis. The effect of MC-IL-2 on Tregs was studied by incubation of splenic T cells with conditioned media obtained from activated BMMCs. Dermatitis was elicited by repeated exposures of mouse ears to oxazolone. MCs in mouse and human skin samples were evaluated by immunostaining. BMMCs released IL-2 in response to IL-33, and IL-2 production was further enhanced by concomitant FcεRI activation. The effect of IL-33 was mediated by activation of the MAPK family members. IL-2 in conditioned media from IL-33 and IgE-stimulated BMMCs led to considerable expansion of Tregs in vitro. IL-33 levels were elevated in oxazolone-challenged ears along with increased numbers of IL-2-expressing MCs. Human skin with chronic inflammation also contained IL-2-expressing MCs that colocalized with IL-33 staining in the dermis. IL-33, in collaboration with IgE, is critical for MC-IL-2 production in allergic skin disease, thus leading to Treg stimulation and suppression of allergic dermatitis. © 2017 John Wiley & Sons Ltd.

Transcription factor EGR-1 suppresses the growth and transformation of human HT-1080 fibrosarcoma cells by induction of transforming growth factor beta 1.

PubMed Central

Liu, C; Adamson, E; Mercola, D

1996-01-01

The early growth response 1 (EGR-1) gene product is a transcription factor with role in differentiation and growth. We have previously shown that expression of exogenous EGR-1 in various human tumor cells unexpectedly and markedly reduces growth and tumorigenicity and, conversely, that suppression of endogenous Egr-1 expression by antisense RNA eliminates protein expression, enhances growth, and promotes phenotypic transformation. However, the mechanism of these effects remained unknown. The promoter of human transforming growth factor beta 1 (TGF-beta 1) contains two GC-rich EGR-1 binding sites. We show that expression of EGR-1 in human HT-1080 fibrosarcoma cells uses increased secretion of biologically active TGF-beta 1 in direct proportion (rPearson = 0.96) to the amount of EGR-1 expressed and addition of recombinant human TGF-beta 1 is strongly growth-suppressive for these cells. Addition of monoclonal anti-TGF-beta 1 antibodies to EGR-1-expressing HT-1080 cells completely reverses the growth inhibitory effects of EGR-1. Reporter constructs bearing the EGR-1 binding segment of the TGF-beta 1 promoter was activated 4- to 6-fold relative to a control reporter in either HT-1080 cells that stably expressed or parental cells cotransfected with an EGR-1 expression vector. Expression of delta EGR-1, a mutant that cannot interact with the corepressors, nerve growth factor-activated factor binding proteins NAB1 and NAB2, due to deletion of the repressor domain, exhibited enhanced transactivation of 2- to 3.5-fold over that of wild-type EGR-1 showing that the reporter construct reflected the appropriate in vivo regulatory context. The EGR-1-stimulated transactivation was inhibited by expression of the Wilms tumor suppressor, a known specific DNA-binding competitor. These results indicate that EGR-1 suppresses growth of human HT-1080 fibrosarcoma cells by induction of TGF-beta 1. Images Fig. 1 Fig. 5 PMID:8876223

AMPK Re-Activation Suppresses Hepatic Steatosis but its Downregulation Does Not Promote Fatty Liver Development.

PubMed

Boudaba, Nadia; Marion, Allison; Huet, Camille; Pierre, Rémi; Viollet, Benoit; Foretz, Marc

2018-02-01

Nonalcoholic fatty liver disease is a highly prevalent component of disorders associated with disrupted energy homeostasis. Although dysregulation of the energy sensor AMP-activated protein kinase (AMPK) is viewed as a pathogenic factor in the development of fatty liver its role has not been directly demonstrated. Unexpectedly, we show here that liver-specific AMPK KO mice display normal hepatic lipid homeostasis and are not prone to fatty liver development, indicating that the decreases in AMPK activity associated with hepatic steatosis may be a consequence, rather than a cause, of changes in hepatic metabolism. In contrast, we found that pharmacological re-activation of downregulated AMPK in fatty liver is sufficient to normalize hepatic lipid content. Mechanistically, AMPK activation reduces hepatic triglyceride content both by inhibiting lipid synthesis and by stimulating fatty acid oxidation in an LKB1-dependent manner, through a transcription-independent mechanism. Furthermore, the effect of the antidiabetic drug metformin on lipogenesis inhibition and fatty acid oxidation stimulation was enhanced by combination treatment with small-molecule AMPK activators in primary hepatocytes from mice and humans. Overall, these results demonstrate that AMPK downregulation is not a triggering factor in fatty liver development but in contrast, establish the therapeutic impact of pharmacological AMPK re-activation in the treatment of fatty liver disease. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

AKT-induced PKM2 phosphorylation signals for IGF-1-stimulated cancer cell growth

PubMed Central

Park, Young Soo; Kim, Dong Joon; Koo, Han; Jang, Se Hwan; You, Yeon-Mi; Cho, Jung Hee; Yang, Suk-Jin; Yu, Eun Sil; Jung, Yuri; Lee, Dong Chul; Kim, Jung-Ae; Park, Zee-Yong; Park, Kyung Chan; Yeom, Young Il

2016-01-01

Pyruvate kinase muscle type 2 (PKM2) exhibits post-translational modifications in response to various signals from the tumor microenvironment. Insulin-like growth factor 1 (IGF-1) is a crucial signal in the tumor microenvironment that promotes cell growth and survival in many human cancers. Herein, we report that AKT directly interacts with PKM2 and phosphorylates it at Ser-202, which is essential for the nuclear translocation of PKM2 protein under stimulation of IGF-1. In the nucleus, PKM2 binds to STAT5A and induces IGF-1-stimulated cyclin D1 expression, suggesting that PKM2 acts as an important factor inducing STAT5A activation under IGF-1 signaling. Concordantly, overexpression of STAT5A in cells deficient in PKM2 expression failed to restore IGF-induced growth, whereas reconstitution of PKM2 in PKM2 knockdown cells restored the IGF-induced growth capacity. Our findings suggest a novel role of PKM2 in promoting the growth of cancers with dysregulated IGF/phosphoinositide 3-kinase/AKT signaling. PMID:27340866

The clinical use of regenerative therapy in COPD

PubMed Central

Lipsi, Roberto; Rogliani, Paola; Calzetta, Luigino; Segreti, Andrea; Cazzola, Mario

2014-01-01

Regenerative or stem cell therapy is an emerging field of treatment based on stimulation of endogenous resident stem cells or administration of exogenous stem cells to treat diseases or injury and to replace malfunctioning or damaged tissues. Current evidence suggests that in the lung, these cells may participate in tissue homeostasis and regeneration after injury. Animal and human studies have demonstrated that tissue-specific stem cells and bone marrow-derived cells contribute to lung tissue regeneration and protection, and thus administration of exogenous stem/progenitor cells or humoral factors responsible for the activation of endogenous stem/progenitor cells may be a potent next-generation therapy for chronic obstructive pulmonary disease. The use of bone marrow-derived stem cells could allow repairing and regenerate the damaged tissue present in chronic obstructive pulmonary disease by means of their engraftment into the lung. Another approach could be the stimulation of resident stem cells by means of humoral factors or photobiostimulation. PMID:25548520

Eluted zinc ions stimulate osteoblast differentiation and mineralization in human dental pulp stem cells for bone tissue engineering.

PubMed

Yusa, Kazuyuki; Yamamoto, Osamu; Iino, Mitsuyoshi; Takano, Hiroshi; Fukuda, Masayuki; Qiao, Zhiwei; Sugiyama, Toshihiro

2016-11-01

Zinc is an essential element for proliferation, differentiation and survival in various cell types. In a previous study, we found that zinc ions released from zinc-modified titanium surfaces (eluted zinc ions; EZ) stimulate cell viability, osteoblast marker gene expression and calcium deposition in human bone marrow-derived mesenchymal cells (hBMCs). The aim of the present study was to investigate the effects of EZ on osteoblast differentiation among dental pulp stem cells (DPSCs) in vitro. In this study, we evaluated the effects of EZ on osteogenesis in DPSCs. Osteoblast and osteoclast marker gene expression was evaluated by real-time PCR. We also evaluated alkaline phosphatase (ALP) staining and calcium deposition. We found that EZ stimulated osteoblast marker gene (type I collagen, alkaline phosphatase (ALP), osteocalcin (OCN) and Runx2) expression, vascular endothelial growth factor A (VEGF-A), and TGF-beta signaling pathway-related gene expression after 7days of incubation. Osteoclastogenesis occurs in a receptor for activated nuclear-factor kappa B ligand (RANKL)/osteoprotegerin (OPG)-independent manner. Real-time PCR analysis revealed that EZ did not affect RANKL or OPG mRNA expression. It was also revealed that EZ induced alkaline phosphatase (ALP) staining and calcium deposition in DPSCs. Collectively, these results demonstrate the potential for clinical application to prospective treatment of bone diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

PubMed

Elattar, Sawsan; Dimri, Manali; Satyanarayana, Ande

2018-03-23

Cachexia is a complex tissue-wasting syndrome characterized by inflammation, hypermetabolism, increased energy expenditure, and anorexia. Browning of white adipose tissue (WAT) is one of the significant factors that contribute to energy wasting in cachexia. By utilizing a cell implantation model, we demonstrate here that the lipid mobilizing factor zinc-α 2 -glycoprotein (ZAG) induces WAT browning in mice. Increased circulating levels of ZAG not only induced lipolysis in adipose tissues but also caused robust browning in WAT. Stimulating WAT progenitors with ZAG recombinant protein or expression of ZAG in mouse embryonic fibroblasts (MEFs) strongly enhanced brown-like differentiation. At the molecular level, ZAG stimulated peroxisome proliferator-activated receptor γ (PPARγ) and early B cell factor 2 expression and promoted their recruitment to the PR/SET domain 16 (Prdm16) promoter, leading to enhanced expression of Prdm16, which determines brown cell fate. In brown adipose tissue, ZAG stimulated the expression of PPARγ and PPARγ coactivator 1α and promoted recruitment of PPARγ to the uncoupling protein 1 (Ucp1) promoter, leading to increased expression of Ucp1. Overall, our results reveal a novel function of ZAG in WAT browning and highlight the targeting of ZAG as a potential therapeutic application in humans with cachexia.-Elattar, S., Dimri, M., Satyanarayana, A. The tumor secretory factor ZAG promotes white adipose tissue browning and energy wasting.

Tumour Necrosis Factor-alpha (TNF-α) and its soluble receptor type 1 (sTNFR I) in human active and healed leishmaniases.

PubMed

Nateghi Rostami, M; Seyyedan Jasbi, E; Khamesipour, A; Mohammadi, A M

2016-04-01

The role of tumour necrosis factor-alpha (TNF-α) is not fully understood in human leishmaniasis. We analysed the alterations in the levels of TNF-α, soluble TNF receptor type 1 (sTNFR I), IL-17 and IL-22 productions in active and healed leishmaniases. Blood samples were collected from volunteers with active cutaneous leishmaniasis (ACL), the same subjects after lesion healing (healed CL = HCL), volunteers with active visceral leishmaniasis (AVL), healed VL (HVL) and healthy controls. Levels of cytokines were titrated on Leishmania Ag-stimulated PBMC culture. The mean level of TNF-α production from stimulated cells was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of sTNFR I production was significantly higher in ACL than controls (P < 0·001) and significantly reduced after treatment in HCL volunteers (P < 0·05). The mean level of IL-22 production in AVL was significantly higher than controls (P < 0·05) and was significantly lower in HVL compared with AVL (P < 0·001) and controls (P < 0·05). The levels of TNF-α (P = 0·0025) and sTNFR I (P < 0·01) productions from PBMCs showed significant decreasing trend after treatment in each CL volunteer. Reduction in TNF-α is associated with clinical response to treatment and healing of CL lesions due to L. major. © 2016 John Wiley & Sons Ltd.

A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

PubMed Central

Thyer, Lynda; Ward, Emma; Smith, Rodney; Fiore, Maria Giulia; Magherini, Stefano; Branca, Jacopo J. V.; Morucci, Gabriele; Gulisano, Massimo; Ruggiero, Marco; Pacini, Stefania

2013-01-01

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects. PMID:23857228

Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells.

PubMed

Hayashida, Tomoko; Decaestecker, Mark; Schnaper, H William

2003-08-01

Transforming growth factor beta (TGF-beta) stimulates renal cell fibrogenesis by a poorly understood mechanism. Previously, we suggested a synergy between TGF-beta1 activated extracellular signal-regulated kinase (ERK) and Smad signaling in collagen production by human glomerular mesangial cells. In a heterologous DNA binding transcription assay, biochemical or dominant-negative ERK blockade reduced TGF-beta1 induced Smad3 activity. Total serine phosphorylation of Smad2/3, but not phosphorylation of the C-terminal SS(P)XS(P) motif, was decreased by pretreatment with the MEK/ERK inhibitors, PD98059 (10 microM) or U0126 (25 microM). This effect was not seen in the mouse mammary epithelial NMuMG cell line, indicating that ERK-dependent activation of Smad2/3 occurs only in certain cell types. TGF-beta stimulated phosphorylation of an expressed Smad3A construct, with a mutated C-terminal SS(P)XS(P) motif, was reduced by a MEK/ERK inhibitor. In contrast, MEK/ERK inhibition did not affect phosphorylation of a Smad3 construct mutated at consensus phosphorylation sites in the linker region (Smad3EPSM). Constitutively active MEK (caMEK) induced alpha2(I) collagen promoter activity, an effect blocked by co-transfected Smad3EPSM, but not Smad3A. The effects of caMEK and TGF-beta1 on collagen promoter activity were additive. These results indicate that ERK-dependent R-Smad linker region phosphorylation enhances collagen I synthesis and imply positive cross talk between the ERK and Smad pathways in human mesangial cells.

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Besemer, J.; Hujber, A.; Kuhn, B.

1989-10-15

The interaction of {sup 125}I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4{degree}C to pH 3 resistance at 37{degree}C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37{degree}C led to nonlinearmore » curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.« less

Cytotoxic Effects of Re-Activated Lunar Dust Stimulant on Human Lung Cells

NASA Technical Reports Server (NTRS)

Upadhyaya, Krishna

2009-01-01

Lunar dust has been of significant concern due to various problems observed on the Apollo missions. Reports from astronauts have shown that the dust may have caused eye and nasal irritation as well as possible hay fever like symptoms. As NASA hopes to go to the Moon within the next few years, we hope to understand the possible toxic effects the dust might have. In these studies, we are looking at the effect of "re-activated" lunar dust stimulant on human bronchial cells. A simple grinding analog as a method of simulating micrometeorite crushing on the moon is used to "activate" the dust stimulant, i.e. capable of producing hydroxyl radicals. These radicals could then interact with human cells and may lead to a loss in membrane integrity and cell death. (Castranova, 1994) Cells are exposed to the dust for 6 and 24 hour intervals to assess cytotoxicity. Cytotoxicity is measured by looking at the production of inflammatory cytokines. Cells are exposed to ground and unground stimulant and compared to cytokine production from cells exposed to quartz which have a known toxicity. Here we look at the cytotoxicity of the lunar dust stimulant relative to quartz by measuring the production of inflammatory cytokines.

Production of cytokines and stimulation of resistance to viral infection in human leukocytes by Scutellaria baicalensis flavones.

PubMed

Błach-Olszewska, Zofia; Jatczak, Bogna; Rak, Anna; Lorenc, Maria; Gulanowski, Bogdan; Drobna, Agnieszka; Lamer-Zarawska, Eliza

2008-09-01

Extracts of Scutellaria baicalensis display a wide spectrum of antiviral activity. It was of great interest to check the effect of baicalein and wogonin preparations on two important mechanisms of innate immunity: the secretion of cytokines and the natural resistance of human leukocytes to viral infection. To study the effect of S. baicalensis extracts on interferons (IFNs), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL) production and virus replication, uninfected and vesicular stomatitis virus (VSV)-infected human peripheral blood leukocytes (PBLs) were used. Four pulverized preparations obtained from roots of Scutellaria and a Sigma-Aldrich preparation of purified baicalein were used in the study. RPMI extracts containing different amounts of baicalein and wogonin were used to study the effect on VSV replication in PBLs. PBLs express ex vivo individually differentiated cytokine-dependent resistance/innate immunity to viral infections. The degree of resistance was estimated on the basis of VSV replication in PBLs. The results obtained indicate that baicalein- and wogonin-containing extracts modulate cytokine production, that is inhibit IFN-alpha and IFN-gamma and stimulate TNF-alpha and IL (IL-12, IL-10) production. They also augment the resistance of PBLs to VSV. Extract from S. baicalensis containing baicalein and wogonin regulates the innate antiviral immunity by modulation of cytokine production and stimulation of human leukocyte resistance.

Quantification of growth factor signaling and pathway cross talk by live-cell imaging.

PubMed

Gross, Sean M; Rotwein, Peter

2017-03-01

Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor-receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras-Raf-Mek-ERK and phosphatidylinositol (PI) 3-kinase-Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways. Copyright © 2017 the American Physiological Society.

Parthenogenic Blastocysts Derived from Cumulus-Free In Vitro Matured Human Oocytes

PubMed Central

McElroy, Sohyun L.; Byrne, James A.; Chavez, Shawn L.; Behr, Barry; Hsueh, Aaron J.; Westphal, Lynn M.; Reijo Pera, Renee A.

2010-01-01

Background Approximately 20% of oocytes are classified as immature and discarded following intracytoplasmic sperm injection (ICSI) procedures. These oocytes are obtained from gonadotropin-stimulated patients, and are routinely removed from the cumulus cells which normally would mature the oocytes. Given the ready access to these human oocytes, they represent a potential resource for both clinical and basic science application. However culture conditions for the maturation of cumulus-free oocytes have not been optimized. We aimed to improve maturation conditions for cumulus-free oocytes via culture with ovarian paracrine/autocrine factors identified by single cell analysis. Methodology/Principal Finding Immature human oocytes were matured in vitro via supplementation with ovarian paracrine/autocrine factors that were selected based on expression of ligands in the cumulus cells and their corresponding receptors in oocytes. Matured oocytes were artificially activated to assess developmental competence. Gene expression profiles of parthenotes were compared to IVF/ICSI embryos at morula and blastocyst stages. Following incubation in medium supplemented with ovarian factors (BDNF, IGF-I, estradiol, GDNF, FGF2 and leptin), a greater percentage of oocytes demonstrated nuclear maturation and subsequently, underwent parthenogenesis relative to control. Similarly, cytoplasmic maturation was also improved as indicated by development to blastocyst stage. Parthenogenic blastocysts exhibited mRNA expression profiles similar to those of blastocysts obtained after IVF/ICSI with the exception for MKLP2 and PEG1. Conclusions/Significance Human cumulus-free oocytes from hormone-stimulated cycles are capable of developing to blastocysts when cultured with ovarian factor supplementation. Our improved IVM culture conditions may be used for obtaining mature oocytes for clinical purposes and/or for derivation of embryonic stem cells following parthenogenesis or nuclear transfer. PMID:20539753

Eukaryotic Translation Initiation Factor 4E Is a Feed-Forward Translational Coactivator of Transforming Growth Factor β Early Protransforming Events in Breast Epithelial Cells

PubMed Central

Decarlo, Lindsey; Mestel, Celine; Barcellos-Hoff, Mary-Helen

2015-01-01

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed early in breast cancers in association with disease progression and reduced survival. Much remains to be understood regarding the role of eIF4E in human cancer. We determined, using immortalized human breast epithelial cells, that elevated expression of eIF4E translationally activates the transforming growth factor β (TGF-β) pathway, promoting cell invasion, a loss of cell polarity, increased cell survival, and other hallmarks of early neoplasia. Overexpression of eIF4E is shown to facilitate the selective translation of integrin β1 mRNA, which drives the translationally controlled assembly of a TGF-β receptor signaling complex containing α3β1 integrins, β-catenin, TGF-β receptor I, E-cadherin, and phosphorylated Smad2/3. This receptor complex acutely sensitizes nonmalignant breast epithelial cells to activation by typically substimulatory levels of activated TGF-β. TGF-β can promote cellular differentiation or invasion and transformation. As a translational coactivator of TGF-β, eIF4E confers selective mRNA translation, reprogramming nonmalignant cells to an invasive phenotype by reducing the set point for stimulation by activated TGF-β. Overexpression of eIF4E may be a proinvasive facilitator of TGF-β activity. PMID:25986608

Investigation of the cortical activation by touching fabric actively using fingers.

PubMed

Wang, Q; Yu, W; He, N; Chen, K

2015-11-01

Human subjects can tactually estimate the perception of touching fabric. Although many psychophysical and neurophysiological experiments have elucidated the peripheral neural mechanisms that underlie fabric hand estimation, the associated cortical mechanisms are not well understood. To identify the brain regions responsible for the tactile stimulation of fabric against human skin, we used the technology of functional magnetic resonance imaging (fMRI), to observe brain activation when the subjects touched silk fabric actively using fingers. Consistent with previous research about brain cognition on sensory stimulation, large activation in the primary somatosensory cortex (SI), the secondary somatosensory cortex (SII) and moto cortex, and little activation in the posterior insula cortex and Broca's Area were observed when the subjects touched silk fabric. The technology of fMRI is a promising tool to observe and characterize the brain cognition on the tactile stimulation of fabric quantitatively. The intensity and extent of activation in the brain regions, especially the primary somatosensory cortex (SI) and the secondary somatosensory cortex (SII), can represent the perception of stimulation of fabric quantitatively. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Non-specific factor enhancement of human in vitro antigen-dependent antibody synthesis: role of B cell activation and T cell help.

PubMed Central

Brenner, M K; North, M E; Chadda, H R; Farrant, J

1984-01-01

Lectin-free supernatants obtained from PWM-stimulated lymphocytes, enable B cells to proliferate and secrete immunoglobulin. Both functions are augmented by the addition of irradiated T cells. In the presence of antigen, these supernatants also enhance specific anti-tetanus toxoid antibody production. The components of the supernatant responsible for these activities have a molecular weight between 30,000 and 60,000, and have the characteristics of non-specific factors: they are genetically unrestricted, and do not bind to either antigen or anti-DR affinity columns. There is no evidence that the partial T dependency of these factors is an indication that their target is a T cell. Instead, T cells appear necessary to move the B cell into a state of activation in which it becomes responsive to the factor. Alternative activation signals such as Staph. A. Cowan can substitute for T cell help in the proliferative response, but not for immunoglobulin or antibody synthesis. The implications of these results for the approaches used to detect and classify B cell growth factors are discussed. PMID:6608488

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

PubMed

Anzinger, Joshua J; Chang, Janet; Xu, Qing; Buono, Chiara; Li, Yifu; Leyva, Francisco J; Park, Bum-Chan; Greene, Lois E; Kruth, Howard S

2010-10-01

To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

TGF-β Affects the Differentiation of Human GM-CSF+ CD4+ T Cells in an Activation- and Sodium-Dependent Manner.

PubMed

Éliás, Szabolcs; Schmidt, Angelika; Kannan, Venkateshan; Andersson, John; Tegnér, Jesper

2016-01-01

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is involved in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis. However, the environmental cues promoting differentiation of GM-CSF producing T cells are unclear. Herein, we performed a broad experimental screening of cytokines and data-driven analysis assessing their ability to induce human GM-CSF + CD4 + T cells and their subpopulations. TGF-β was discovered to induce GM-CSF production independently of proliferation and IL-2 signaling including STAT5. In contrast, IL-6 and IL-23 decreased GM-CSF production. On the population level, GM-CSF induction was highly correlated with expression of FOXP3 across cytokine stimulations but not with that of IL-17. However, on single-cell level GM-CSF and IFN-γ expression were most correlated, independently of the cytokine environment. Importantly, under low sodium conditions in the medium or upon stimulation with plate-bound instead of bead-bound anti-CD3 and anti-CD28 antibodies, the effects of TGF-β on GM-CSF, but not on FOXP3, were reversed. Our analysis indicates a novel role for TGF-β in generating GM-CSF + subsets of human CD4 + T cells. These results are important for understanding of autoimmune disease and therapeutic considerations.

Interleukin 1 β-induced SMAD2/3 linker modifications are TAK1 dependent and delay TGFβ signaling in primary human mesenchymal stem cells.

PubMed

van den Akker, Guus G; van Beuningen, Henk M; Vitters, Elly L; Koenders, Marije I; van de Loo, Fons A; van Lent, Peter L; Blaney Davidson, Esmeralda N; van der Kraan, Peter M

2017-12-01

Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFβ) signaling. TGFβ binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 β (IL1β) on SMAD2/3 signaling dynamics and post-translational modifications. Co-stimulation of MSC with TGFβ and IL1β did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA 12 -luciferase reporter construct, was enhanced by co-stimulation of TGFβ and IL1β compared to TGFβ alone. Furthermore, IL1β stimulation induced CAGA 12 -luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFβ signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFβ, IL1β or TGFβ and IL1β stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1β-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFβ. Co-stimulation with both factors resulted in a similar kinetic profile as TGFβ alone. Nevertheless, IL1β did subtly alter TGFβ-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFβ target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1β on TGFβ signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1β-induced SMAD2 linker modifications and CAGA 12 -luciferase activity. TGFβ and IL1β signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFβ target genes were repressed by IL1β independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1β-induced TGFβ signal modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

12,13-diHOME: An Exercise-Induced Lipokine that Increases Skeletal Muscle Fatty Acid Uptake.

PubMed

Stanford, Kristin I; Lynes, Matthew D; Takahashi, Hirokazu; Baer, Lisa A; Arts, Peter J; May, Francis J; Lehnig, Adam C; Middelbeek, Roeland J W; Richard, Jeffrey J; So, Kawai; Chen, Emily Y; Gao, Fei; Narain, Niven R; Distefano, Giovanna; Shettigar, Vikram K; Hirshman, Michael F; Ziolo, Mark T; Kiebish, Michael A; Tseng, Yu-Hua; Coen, Paul M; Goodyear, Laurie J

2018-05-01

Circulating factors released from tissues during exercise have been hypothesized to mediate some of the health benefits of regular physical activity. Lipokines are circulating lipid species that have recently been reported to affect metabolism in response to cold. Here, lipidomics analysis revealed that a bout of moderate-intensity exercise causes a pronounced increase in the circulating lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) in male, female, young, old, sedentary, and active human subjects. In mice, both a single bout of exercise and exercise training increased circulating 12,13-diHOME and surgical removal of brown adipose tissue (BAT) negated the increase in 12,13-diHOME, suggesting that BAT is the tissue source for exercise-stimulated 12,13-diHOME. Acute 12,13-diHOME treatment of mice in vivo increased skeletal muscle fatty acid uptake and oxidation, but not glucose uptake. These data reveal that lipokines are novel exercise-stimulated circulating factors that may contribute to the metabolic changes that occur with physical exercise. Copyright © 2018 Elsevier Inc. All rights reserved.

Lemongrass (Cymbopogon flexuosus) essential oil demonstrated anti-inflammatory effect in pre-inflamed human dermal fibroblasts.

PubMed

Han, Xuesheng; Parker, Tory L

2017-06-01

Lemongrass ( Cymbopogon flexuosus ) essential oil (LEO), which has citral as its main component, has exhibited anti-inflammatory effect in both animal and human cells. In this study, we evaluated the anti-inflammatory activity of a commercially available LEO in pre-inflamed human dermal fibroblasts. We first studied the impact of LEO on 17 protein biomarkers that are critically associated with inflammation and tissue remodeling. LEO significantly inhibited production of the inflammatory biomarkers vascular cell adhesion molecule 1 (VCAM-1), interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell alpha chemoattractant (I-TAC), and monokine induced by gamma interferon (MIG); decreased levels of the tissue remodeling biomarkers collagen-I and III, epidermal growth factor receptor (EGFR), and plasminogen activator inhibitor (PAI-1); and inhibited the immunomodulatory biomarker macrophage colony-stimulating factor (M-CSF). Furthermore, we studied the impact of LEO on genome-wide gene expression profiles. LEO significantly modulated global gene expression and robustly impacted signaling pathways, many of which are critical for inflammation and tissue remodeling processes. This study provides the first evidence of the anti-inflammatory activity of LEO in human skin cells and indicates that it is a good therapeutic candidate for treating inflammatory conditions of the skin.

A concise review of testosterone and bone health

PubMed Central

Mohamad, Nur-Vaizura; Soelaiman, Ima-Nirwana; Chin, Kok-Yong

2016-01-01

Osteoporosis is a condition causing significant morbidity and mortality in the elderly population worldwide. Age-related testosterone deficiency is the most important factor of bone loss in elderly men. Androgen can influence bone health by binding to androgen receptors directly or to estrogen receptors (ERs) indirectly via aromatization to estrogen. This review summarized the direct and indirect effects of androgens on bone derived from in vitro, in vivo, and human studies. Cellular studies showed that androgen stimulated the proliferation of preosteoblasts and differentiation of osteoblasts. The converted estrogen suppressed osteoclast formation and resorption activity by blocking the receptor activator of nuclear factor k-B ligand pathway. In animal studies, activation of androgen and ERα, but not ERβ, was shown to be important in acquisition and maintenance of bone mass. Human epidemiological studies demonstrated a significant relationship between estrogen and testosterone in bone mineral density and fracture risk, but the relative significance between the two remained debatable. Human experimental studies showed that estrogen was needed in suppressing bone resorption, but both androgen and estrogen were indispensable for bone formation. As a conclusion, maintaining optimal level of androgen is essential in preventing osteoporosis and its complications in elderly men. PMID:27703340

In Vitro Human Umbilical Vein Endothelial Cells Response to Ionic Dissolution Products from Lithium-Containing 45S5 Bioactive Glass

PubMed Central

Haro Durand, Luis A.; Vargas, Gabriela E.; Vera-Mesones, Rosa; Baldi, Alberto; Zago, María P.; Fanovich, María A.; Boccaccini, Aldo R.; Gorustovich, Alejandro

2017-01-01

Since lithium (Li+) plays roles in angiogenesis, the localized and controlled release of Li+ ions from bioactive glasses (BGs) represents a promising alternative therapy for the regeneration and repair of tissues with a high degree of vascularization. Here, microparticles from a base 45S5 BG composition containing (wt %) 45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5, in which Na2O was partially substituted by 5% Li2O (45S5.5Li), were obtained. The results demonstrate that human umbilical vein endothelial cells (HUVECs) have greater migratory and proliferative response and ability to form tubules in vitro after stimulation with the ionic dissolution products (IDPs) of the 45S5.5Li BG. The results also show the activation of the canonical Wnt/β-catenin pathway and the increase in expression of proangiogenic cytokines insulin like growth factor 1 (IGF1) and transforming growth factor beta (TGFβ). We conclude that the IDPs of 45S5.5Li BG would act as useful inorganic agents to improve tissue repair and regeneration, ultimately stimulating HUVECs behavior in the absence of exogenous growth factors. PMID:28773103

ROS is Required for Alternatively Activated Macrophage Differentiation | Center for Cancer Research

Cancer.gov

Macrophages are key regulators in host inflammatory responses. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are responsible for inducing macrophage differentiation from monocytes. GM-CSF or M-CSF-differentiated macrophages can be further differentiated, or polarized, to more specialized cells. Classically activated,

Cinnamophilin isolated from Cinnamomum philippinense protects against collagen degradation in human chondrocytes.

PubMed

Lu, Yung-Chang; Hsiao, George; Lin, Kuan-Hung; Hsieh, Ming-Shium; Jayakumar, Thanasekaran; Wu, Tian-Shung; Sheu, Joen-Rong

2013-06-01

To investigate the therapeutic potential of naturally occurring cinnamophilin against cartilage degradation and its action mechanisms, its effects on matrix metalloproteinase (MMP)-1 and -13 induction were examined in the human SW1353 chondrocytic cell line. Human chondrocytes (SW1353) were stimulated with interleukin (IL)-1β, and then mitogen-activated protein kinase (MAPK) and c-Jun activations, inhibitory κB-α (IκB-α) degradation, and MMP-1, and 13 expressions were assayed by a Western blot analysis. Cinnamophilin strongly inhibited MMP-1 and -13 induction in IL-1β-treated (30 ng/mL) SW1353 cells in a concentration-dependent manner, and it also reduced MAPK family members including extracellular signal-regulated kinase (ERK), p38 MAPKs, and c-Jun N-terminal kinase. Moreover, nuclear factor (NF)-κB signaling activation through IκB-α degradation, IκB kinase (IKK)-α/β, and p-65 phosphorylation was restored by cinnamophilin upon IL-1β stimulation. Importantly, results showed that IL-1β-induced activation of phosphorylated (p)-c-Jun in chondrocytes was significantly inhibited by cinnamophilin. These results indicate that cinnamophilin inhibited MMP-1 and -13 expressions in IL-1β-treated chondrocytes through either NF-κB or ERK/p38 MAPK downregulation and/or suppressing p-c-Jun pathways. Furthermore, these findings suggest that cinnamophilin may have the potential for chondroprotection against collagen matrix breakdown in cartilage of diseased tissues such as those found in arthritic disorders. Copyright © 2012 John Wiley & Sons, Ltd.

Anaplastic lymphoma kinase is expressed in different subtypes of human breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Pinera, Pablo; Chang, Y.; Astudillo, A.

2007-06-29

Pleiotrophin (PTN, Ptn) is an 18 kDa cytokine expressed in human breast cancers. Since inappropriate expression of Ptn stimulates progression of breast cancer in transgenic mice and a dominant negative PTN reverses the transformed phenotype of human breast cancer cells that inappropriately express Ptn, it is suggested that constitutive PTN signaling in breast cancer cells that inappropriately express Ptn activates pathways that promote a more aggressive breast cancer phenotype. Pleiotrophin signals by inactivating its receptor, the receptor protein tyrosine phosphatase (RPTP){beta}/{zeta}, and, recently, PTN was found to activate anaplastic lymphoma kinase (ALK) through the PTN/RPTP{beta}/{zeta} signaling pathway in PTN-stimulated cells,more » not through a direct interaction of PTN with ALK and thus not through the PTN-enforced dimerization of ALK. Since full-length ALK is activated in different malignant cancers and activated ALK is a potent oncogenic protein, we examined human breast cancers to test the possibility that ALK may be expressed in breast cancers and potentially activated through the PTN/RPTP{beta}/{zeta} signaling pathway; we now demonstrate that ALK is strongly expressed in different histological subtypes of human breast cancer; furthermore, ALK is expressed in both nuclei and cytoplasm and, in the 'dotted' pattern characteristic of ALK fusion proteins in anaplastic large cell lymphoma. This study thus supports the possibility that activated ALK may be important in human breast cancers and potentially activated either through the PTN/RPTP{beta}/{zeta} signaling pathway, or, alternatively, as an activated fusion protein to stimulate progression of breast cancer in humans.« less

Quantification of growth factor signaling and pathway cross talk by live-cell imaging

PubMed Central

Gross, Sean M.

2017-01-01

Peptide growth factors stimulate cellular responses through activation of their transmembrane receptors. Multiple intracellular signaling cascades are engaged following growth factor–receptor binding, leading to short- and long-term biological effects. Each receptor-activated signaling pathway does not act in isolation but rather interacts at different levels with other pathways to shape signaling networks that are distinctive for each growth factor. To gain insights into the specifics of growth factor-regulated interactions among different signaling cascades, we developed a HeLa cell line stably expressing fluorescent live-cell imaging reporters that are readouts for two major growth factor-stimulated pathways, Ras–Raf–Mek–ERK and phosphatidylinositol (PI) 3-kinase–Akt. Incubation of cells with epidermal growth factor (EGF) resulted in rapid, robust, and sustained ERK signaling but shorter-term activation of Akt. In contrast, hepatocyte growth factor induced sustained Akt signaling but weak and short-lived ERK activity, and insulin-like growth factor-I stimulated strong long-term Akt responses but negligible ERK signaling. To address potential interactions between signaling pathways, we employed specific small-molecule inhibitors. In cells incubated with EGF or platelet-derived growth factor-AA, Raf activation and the subsequent stimulation of ERK reduced Akt signaling, whereas Mek inhibition, which blocked ERK activation, enhanced Akt and turned transient effects into sustained responses. Our results reveal that individual growth factors initiate signaling cascades that vary markedly in strength and duration and demonstrate in living cells the dramatic effects of cross talk from Raf and Mek to PI 3-kinase and Akt. Our data further indicate how specific growth factors can encode distinct cellular behaviors by promoting complex interactions among signaling pathways. PMID:28100485

The action of sennosides and related compounds on human colon and rectum 1

PubMed Central

Hardcastle, J. D.; Wilkins, J. L.

1970-01-01

The direct action of intraluminal senna and related compounds on the human colon and rectum has been investigated. Motility was recorded by balloon kymography with recording units inserted into well established transverse colostomies or into the rectum. The motility of the colon was not changed by intraluminal senna glycosides but the introduction of senna previously incubated with faeces or Esch. coli stimulated the colon to peristalt. The peristalsis was similar to that stimulated by rheinanthrone, an oxanthrone produced by chemical hydrolysis and reduction of senna. Both activated senna and rheinanthrone appeared to act in the colon by contact stimulation. No peristaltic response was stimulated in the rectum, either with activated senna or with rheinanthrone. PMID:4929273

The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase.

PubMed Central

Cavigelli, M; Li, W W; Lin, A; Su, B; Yoshioka, K; Karin, M

1996-01-01

Trivalent arsenic (As3+) is highly carcinogenic, but devoid of known mutagenic activity. Therefore, it is likely to act as a tumor promoter. To understand the molecular basis for the tumor-promoting activity of As3+, we examined its effect on transcription factor AP-1, whose activity is stimulated by several other tumor promoters. We found that As3+, but not As5+, which is toxic but not carcinogenic, is a potent stimulator of AP-1 transcriptional activity and an efficient inducer of c-fos and c-jun gene expression. Induction of c-jun and c-fos transcription by As3+ correlates with activation of Jun kinases (JNKs) and p38/Mpk2, which phosphorylate transcription factors that activate these immediate early genes. No effect on ERK activity was observed. As5+, on the other hand, had a negligible effect on JNK or p38/Mpk2 activity. Biochemical analysis and co-transfection experiments strongly suggest that the primary mechanism by which As3+ stimulates JNK activity involves the inhibition of a constitutive dual-specificity JNK phosphatase. This phosphatase activity appears to be responsible for maintaining low basal JNK activity in non-stimulated cells and its inhibition may lead to tumor promotion through induction of proto-oncogenes such as c-jun and c-fos, and stimulation of AP-1 activity. The same phosphatase may also regulate p38/Mpk2 activity. Images PMID:8947050

The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

PubMed Central

Hoxhaj, Gerta; Caddye, Edward; Najafov, Ayaz; Houde, Vanessa P; Johnson, Catherine; Dissanayake, Kumara; Toth, Rachel; Campbell, David G; Prescott, Alan R; MacKintosh, Carol

2016-01-01

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1. DOI: http://dx.doi.org/10.7554/eLife.12278.001 PMID:27244671

Human papillomavirus oncogenic E6 protein regulates human β-defensin 3 (hBD3) expression via the tumor suppressor protein p53

PubMed Central

Yue, Hong; Wang, Liming; Jin, Jessica; Ghosh, Santosh K.; Kawsar, Hameem I.; Zender, Chad; Androphy, Elliot J.; Weinberg, Aaron; McCormick, Thomas S.; Jin, Ge

2016-01-01

Human β-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer. PMID:27034006

Transcription factor specificity protein 1 modulates TGFβ1/Smad signaling to negatively regulate SIGIRR expression by human M1 macrophages stimulated with substance P.

PubMed

Yamaguchi, Rui; Sakamoto, Arisa; Yamaguchi, Reona; Haraguchi, Misa; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

2018-08-01

The stimuli inducing expression of single immunoglobulin IL-1-related receptor (SIGIRR) and the relevant regulatory mechanisms are not well defined. Transforming growth factor β1 (TGFβ1) delays internalization of neurokinin-1 receptor (NK1R) and subsequently enhances cellular signaling. This study investigated the effect of TGFβ1 on SIGIRR protein production by human M1 macrophages in response to stimulation with substance P (SP). SP caused upregulation of SIGIRR expression in a concentration-dependent manner, whereas aprepitant (an NK1R inhibitor) blunted this response. Silencing p38γMAPK or TAK-1 partially attenuated the response to SP stimulation, while TGFβ1/2/3 siRNA dramatically diminished it. SP induced much greater SIGIRR protein production than either lipopolysaccharide (a TLR4 agonist) or resiquimod (a TLR7/8 agonist). Unexpectedly, silencing of transcription factor specificity protein 1 (Sp1) led to significant upregulation of SIGIRR expression after SP stimulation, while KLF2 siRNA only partially enhanced it and Fli-1 siRNA reduced it. SP also upregulated TGFβ1 expression, along with a corresponding increase of SIGIRR protein, whereas silencing TGFβ1/2/3 blunted these responses. Sp1 siRNA or mithramycin (a gene-selective Sp1 inhibitor) significantly enhanced the expression of TGFβ1 and SIGIRR by macrophages after SP stimulation. Importantly, this effect of Sp1 siRNA on TGFβ1 and SIGIRR was blunted by siRNA for Smad2, Smad3, or Smad4, but not by TAK-1 siRNA. Next, we investigated the influence of transcription factor cross-talk on SIGIRR expression in response to SP. Co-transfection of macrophages with Sp1 siRNA and C/EBPβ or TIF1β siRNA attenuated the upregulation of SIGIRR by SP, while a combination of Sp1 siRNA and Fli-1 siRNA dramatically diminished it. In conclusion, TGFβ1 may be an intermediary between SP/NK1R activation and SIGIRR expression in Sp1 siRNA-transfected macrophages. In addition, Sp1 modulates TGFβ1/Smad signaling and negatively regulates SIGIRR protein production by macrophages after SP stimulation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System

PubMed Central

Ravindra, Kodihalli C.; Large, Emma; Young, Carissa L.; Rivera-Burgos, Dinelia; Yu, Jiajie; Cirit, Murat; Hughes, David J.; Wishnok, John S.; Lauffenburger, Douglas A.; Griffith, Linda G.

2017-01-01

In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro–in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography–tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell–mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1β, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1β, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans. PMID:28450578

Long-term forskolin stimulation induces AMPK activation and thereby enhances tight junction formation in human placental trophoblast BeWo cells.

PubMed

Egawa, M; Kamata, H; Kushiyama, A; Sakoda, H; Fujishiro, M; Horike, N; Yoneda, M; Nakatsu, Y; Ying, Guo; Jun, Zhang; Tsuchiya, Y; Takata, K; Kurihara, H; Asano, T

2008-12-01

BeWo cells, derived from human choriocarcinoma, have been known to respond to forskolin or cAMP analogues by differentiating into multinucleated cells- like syncytiotrophoblasts on the surfaces of chorionic villi of the human placenta. In this study, we demonstrated that long-term treatment with forskolin enhances the tight junction (TJ) formation in human placental BeWo cells. Interestingly, AMPK activation and phosphorylation of acetyl-CoA carboxylase (ACC), a molecule downstream from AMPK, were induced by long-term incubation (>12h) with forskolin, despite not being induced by acute stimulation with forskolin. In addition, co-incubation with an AMPK inhibitor, compound C, as well as overexpression of an AMPK dominant negative mutant inhibited forskolin-induced TJ formation. Thus, although the molecular mechanism underlying AMPK activation via the forskolin stimulation is unclear, the TJ formation induced by forskolin is likely to be mediated by the AMPK pathway. Taking into consideration that TJs are present in the normal human placenta, this mechanism may be important for forming the placental barrier system between the fetal and maternal circulations.

Mangiferin attenuates the symptoms of dextran sulfate sodium-induced colitis in mice via NF-κB and MAPK signaling inactivation

PubMed Central

Dou, Wei; Zhang, Jingjing; Ren, Gaiyan; Ding, Lili; Sun, Aning; Deng, Chao; Wu, Xiaojun; Wei, Xiaohui; Mani, Sridhar; Wang, Zhengtao

2015-01-01

Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal (GI) tract, and currently no curative treatment available. Mangiferin, a natural glucosylxanthone mainly from the fruit, leaves and stem bark of the mango tree, has strong anti-inflammatory activity. We sought to investigate whether mangiferin attenuates inflammation in a mouse model of chemically induced IBD. Pre-administration of mangiferin significantly attenuated dextran sulfate sodium (DSS)-induced body weight loss, diarrhea, colon shortening and histological injury, which correlated with the decline in the activity of myeloperoxidase (MPO) and the level of tumor necrosis factor-α (TNF-α) in the colon. DSS-induced degradation of inhibitory κBα (IκBα) and the phosphorylation of nuclear factor-kappa B (NF-κB) p65 as well as the mRNA expression of pro-inflammatory mediators (inducible NO synthase (iNOS), intercellular adhesion molecule-1 (ICAM-1), TNF-α, interleukin-1β (IL-1β) and IL-6) in the colon were also downregulated by mangiferin treatment. Additionally, the phosphorylation/activation of DSS-induced mitogen-activated protein kinase (MAPK) proteins was also inhibited by mangiferin treatment. In accordance with the in vivo results, mangiferin exposure blocked TNF-α-stimulated nuclear translocation of NF-κB in RAW264.7 mouse macrophage cells. Transient transfection gene reporter assay performed in TNF-α-stimulated HT-29 human colorectal adenocarcinoma cells indicated that mangiferin inhibits NF-κB transcriptional activity in a dose-dependent manner. The current study clearly demonstrates a protective role for mangiferin in experimental IBD through NF-κB and MAPK signaling inhibition. Since mangiferin is a natural compound with little toxicity, the results may contribute to the effective utilization of mangiferin in the treatment of human IBD. PMID:25194678

Interleukin-1 stimulates zinc uptake by human thymic epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coto, J.A.; Hadden, J.W.

1991-03-15

Thymic epithelial cells (TEC) are known to secrete peptides which influence the differentiation and maturation of T-lymphocytes. These peptides include the thymic hormones thymulin, thymosin-{alpha}1, and thymopoietin. The biological activity of thymulin is dependent on the presence of zinc in an equimolar ratio. The authors have shown that both interleukin-1{alpha}(IL-1{alpha}) and interleukin-1{beta}(IL-1{beta}), which stimulate proliferation of TEC, stimulate the uptake of Zn-65 in-vitro independent of this proliferation. Mitomycin-C was used to inhibit the proliferation of TEC. Two other stimulators of proliferation of TEC, bovine pituitary extract (BPE) and epidermal growth factor (EGF), did not stimulate zinc uptake by the TECmore » independent of proliferation. They have also shown, utilizing in-situ hybridization, that IL-1 and zinc induce metallothionein(MT) mRNA expression in human thymic epithelial cells. The exact role of metallothionein is not clear, but it is thought to be involved in regulation of trace metal metabolism, especially in maintenance of zinc homeostasis. Their current hypothesis is that IL-1 stimulates uptake of zinc into the TEC, followed by its complexing with metallothionein. Zinc is then thought to be transferred from metallothionein to thymulin. Immunostaining, utilizing an antithymulin antibody and a fluoresceinated goat anti-rabbit second antibody, confirms the presence of thymulin in TEC and its dependence on zinc. Upon stimulation, thymulin is then secreted. Known stimulants for thymulin include progesterone, dexamethasone, estradiol, testosterone, and prolactin. None of these secretagogues increase zinc uptake, suggesting the priming of the zinc-thymulin complex is unrelated to the regulation of its secretion.« less

HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine.

PubMed

Asea, A; Kraeft, S K; Kurt-Jones, E A; Stevenson, M A; Chen, L B; Finberg, R W; Koo, G C; Calderwood, S K

2000-04-01

Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-kappaB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1beta, IL-6 and TNF-alpha; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-alpha but not IL-1beta or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

Preferential Expression of PAPP-A in Human Preadipocytes from Omental Fat

PubMed Central

Davidge-Pitts, Caroline; Escande, Carlos J.; Conover, Cheryl A.

2014-01-01

Fat distribution differs between individuals, and those with visceral fat predominance develop metabolic profiles that increase risk of adverse cardiovascular events. This is due, in part, to the proinflammatory state associated with visceral obesity as well as depot-specific adipogenesis. The insulin-like growth factor (IGF) system is important in adipose tissue development and metabolic function. Pregnancy associated plasma protein-A (PAPP-A) is a novel zinc metalloproteinase that regulates local IGF availability. The first aim of this study was to characterize PAPP-A mRNA and protein expression in primary cultures of human preadipocytes isolated from omental, mesenteric and subcutaneous depots. PAPP-A expression was significantly increased in omental preadipocytes compared to mesenteric and subcutaneous preadipocytes. The second aim was to investigate factors regulating PAPP-A expression, focusing on proinflammatory cytokines and resveratrol that have been shown to have negative and positive effects, respectively, on metabolism and diet-induced obesity. Treatment of cultured primary human preadipocytes with tumor necrosis factor (TNF)-α and interleukin (IL) 1-β led to significant increases in PAPP-A expression. Activated pathways mediating cytokine-induced PAPP-A expression include the nuclear factor (NF) κB pathway and the mitogen activated protein kinase (MAPK) family, particularly c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated kinase. Resveratrol, a polyphenol with beneficial cardiometabolic effects, significantly down-regulated PAPP-A expression under basal and stimulated conditions. Resveratrol appeared to mediate its effects on PAPP-A through pathways independent of silent mating type information regulation 2 homolog 1 (SIRT1) and AMP kinase (AMPK) activation. Depot-specific PAPP-A expression in human preadipocytes may contribute to depot-specific function. PMID:24781252

Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation.

PubMed Central

Cavigelli, M; Dolfi, F; Claret, F X; Karin, M

1995-01-01

Growth factors induce c-fos transcription by stimulating phosphorylation of transcription factor TCF/Elk-1, which binds to the serum response element (SRE). Under such conditions Elk-1 could be phosphorylated by the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2. However, c-fos transcription and SRE activity are also induced by stimuli, such as UV irradiation and activation of the protein kinase MEKK1, that cause only an insignificant increase in ERK1/2 activity. However, both of these stimuli strongly activate two other MAPKs, JNK1 and JNK2, and stimulate Elk-1 transcriptional activity and phosphorylation. We find that the JNKs are the predominant Elk-1 activation domain kinases in extracts of UV-irradiated cells and that immunopurified JNK1/2 phosphorylate Elk-1 on the same major sites recognized by ERK1/2, that potentiate its transcriptional activity. Finally, we show that UV irradiation, but not serum or phorbol esters, stimulate translocation of JNK1 to the nucleus. As Elk-1 is most likely phosphorylated while bound to the c-fos promoter, these results suggest that UV irradiation and MEKK1 activation stimulate TCF/Elk-1 activity through JNK activation, while growth factors induce c-fos through ERK activation. Images PMID:8846788

Selective Activation of Resting-State Networks following Focal Stimulation in a Connectome-Based Network Model of the Human Brain

PubMed Central

2016-01-01

Abstract When the brain is stimulated, for example, by sensory inputs or goal-oriented tasks, the brain initially responds with activities in specific areas. The subsequent pattern formation of functional networks is constrained by the structural connectivity (SC) of the brain. The extent to which information is processed over short- or long-range SC is unclear. Whole-brain models based on long-range axonal connections, for example, can partly describe measured functional connectivity dynamics at rest. Here, we study the effect of SC on the network response to stimulation. We use a human whole-brain network model comprising long- and short-range connections. We systematically activate each cortical or thalamic area, and investigate the network response as a function of its short- and long-range SC. We show that when the brain is operating at the edge of criticality, stimulation causes a cascade of network recruitments, collapsing onto a smaller space that is partly constrained by SC. We found both short- and long-range SC essential to reproduce experimental results. In particular, the stimulation of specific areas results in the activation of one or more resting-state networks. We suggest that the stimulus-induced brain activity, which may indicate information and cognitive processing, follows specific routes imposed by structural networks explaining the emergence of functional networks. We provide a lookup table linking stimulation targets and functional network activations, which potentially can be useful in diagnostics and treatments with brain stimulation. PMID:27752540

Detection of the CLOCK/BMAL1 heterodimer using a nucleic acid probe with cycling probe technology.

PubMed

Nakagawa, Kazuhiro; Yamamoto, Takuro; Yasuda, Akio

2010-09-15

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), has enabled rapid acquisition of genomic information. Here we report an analogous technique for the detection of an activated transcription factor, a transcription element-binding assay with fluorescent amplification by apurinic/apyrimidinic (AP) site lysis cycle (TEFAL). This simple amplification assay can detect activated transcription factors by using a unique nucleic acid probe containing a consensus binding sequence and an AP site, which enables the CPT reaction with AP endonuclease. In this article, we demonstrate that this method detects the functional CLOCK/BMAL1 heterodimer via the TEFAL probe containing the E-box consensus sequence to which the CLOCK/BMAL1 heterodimer binds. Using TEFAL combined with immunoassays, we measured oscillations in the amount of CLOCK/BMAL1 heterodimer in serum-stimulated HeLa cells. Furthermore, we succeeded in measuring the circadian accumulation of the functional CLOCK/BMAL1 heterodimer in human buccal mucosa cells. TEFAL contributes greatly to the study of transcription factor activation in mammalian tissues and cell extracts and is a powerful tool for less invasive investigation of human circadian rhythms. 2010 Elsevier Inc. All rights reserved.

The MAP kinase pathway is involved in odontoblast stimulation via p38 phosphorylation.

PubMed

Simon, Stephane; Smith, Anthony J; Berdal, Ariane; Lumley, Philip J; Cooper, Paul R

2010-02-01

We have previously shown that the p38 gene is highly expressed in odontoblasts during active primary dentinogenesis, but is drastically down-regulated as cells become quiescent in secondary dentinogenesis. Based on these observations, we hypothesized that p38 expression might be upregulated, and the protein activated by phosphorylation, when odontoblasts are stimulated such as during tertiary reactionary dentinogenesis. We stimulated immortalized, odontoblast-like MDPC-23 cells, alone or in combination, with heat-inactivated Streptococcus mutans, EDTA-extracted dentine matrix proteins (DMPs), or growth factors, including transforming growth factor (TGF)-beta1, tumor necrosis factor-alpha (TNF-alpha), and adrenomedullin (ADM). We used ELISA to measure the resulting phosphorylation of the p38 protein, as well as its degree of nuclear translocation. Our results suggest that the p38-MAPKinase pathway is activated during odontoblast stimulation in tertiary dentinogenesis by both p38 phosphorylation and enhanced nuclear translocation. Data indicate that odontoblast behaviour therefore potentially recapitulates that during active primary dentinogenesis. Copyright 2010 American Association of Endodontists. All rights reserved.