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Id Subject Object Predicate Lexical cue
T110 0-76 Sentence denotes Insights into the Regulation of TNF-α Production in Human Mononuclear Cells:
T1 0-76 Sentence denotes Insights into the Regulation of TNF-α Production in Human Mononuclear Cells:
T111 77-133 Sentence denotes The Effects of Non-Specific Phosphodiesterase Inhibition
T2 77-133 Sentence denotes The Effects of Non-Specific Phosphodiesterase Inhibition
T3 136-144 Sentence denotes Abstract
T112 145-154 Sentence denotes OBJECTIVE
T4 145-154 Sentence denotes OBJECTIVE
T113 155-404 Sentence denotes The objective of this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells.
T5 155-404 Sentence denotes The objective of this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells.
T2453 172-5223 Sentence denotes this study was to determine the effect of nonspecific phosphodiesterase inhibition on transcription factor activation and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS)-stimulated human mononuclear cells. INTRODUCTION The production of TNF-α following LPS stimulation is one of the key steps in bacterial sepsis and inflammation. The mechanism by which phosphodiesterase inhibition alters TNF-α production in the presence of LPS remains unclear. METHODS Human mononuclear cells were stimulated with LPS (1 μg/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor. Western blotting of phosphorylated cytoplasmic I-κBα, nuclear factor-κB p65 (NF-κB), and nuclear cAMP-response element binding protein (CREB) was performed. DNA binding of NF-κB and CREB was verified by electrophoretic mobility shift assay. TNF-α levels were determined in the supernatant of stimulated cells in the presence and absence Protein kinase A inhibition by an enzyme-linked immunosorbent assay (ELISA). RESULTS PTX was demonstrated to significantly reduce cytoplasmic I-κBα phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-κB. In contrast, PTX markedly enhanced the phosphorylation and DNA binding activity of CREB. Cells concomitantly treated with PTX and LPS secreted similar levels of TNF-α in the presence and absence Protein kinase A inhibition. DISCUSSION The increased level of cAMP that results from phosphodiesterase inhibition affects cytoplasmic and nuclear events, resulting in the attenuation of NF-κB and the activation of CREB transcriptional DNA binding through pathways that are partially Protein kinase A-independent. CONCLUSION PTX-mediated phosphodiesterase inhibition occurs partially through a Protein kinase A-independent pathway and may serve as a useful tool in the attenuation of LPS-induced inflammation. INTRODUCTION The bacterial membrane component lipopolysaccharide (LPS) is capable of initiating phosphorylation and activation of multiple host intracellular protein kinases and transcription factors. Transcription factor activation, which results in the modulation of gene transcription and protein synthesis, is a critical element in the defense mechanism of the host immune system.1,2 LPS-induced transcription factor activation has been shown to be a key regulator of tumor necrosis factor-α (TNF-α) production.3 TNF-α is a potent pro-inflammatory cytokine involved in a wide spectrum of cellular responses. Furthermore, TNF-α synthesis can be attenuated in immune cells exposed to phosphodiesterase (PDE) inhibition after challenge by a variety of pro-inflammatory stimulants.4 The signaling mechanisms affected by PDE inhibition, which ultimately lead to the downregulation of TNF-α production, have not been well characterized in inflammatory cells. Classically, it has been demonstrated that PDE inhibition results in the intracellular accumulation of the second messenger cyclic adenosine-3,5-monophosphate (cAMP) and subsequent activation of Protein kinase A (PKA).5 PKA activation then leads to the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), transmission of signals into the nucleus, and the subsequent modulation of gene transcription.6 This apparently simple linear cascade does not fully explain the mechanism by which an elevation in the intracellular cAMP level exerts wide-ranging effects on multiple cellular functions. There is a growing body of evidence suggesting that cAMP may function through both PKA-dependent and -independent mechanisms.6–8 LPS-induced activation of the transcription factor nuclear factor-κB (NF-κB) has also been the focus of a great deal of research. It has been clearly demonstrated that agents that increase intracellular cAMP also inhibit NF-κB-dependent pro-inflammatory gene transcription, particularly of the TNF-α gene.9 Controversy exists regarding the exact mechanism(s) by which PDE inhibition down-regulates TNF-α production. Possibilities include, but are not limited to, inhibition of NF-κB DNA binding activity, downregulation of NF-κB transcriptional activity, increased CREB activation, and competition between NF-κB and CREB for common co-activators such as CREB binding protein (CBP). It is also not known whether these processes rely solely upon the activation of PKA. Therefore, the objective of the present study is to determine the effects of nonspecific PDE inhibition with 1-[5-oxohexyl]-3,7-dimethylxanthine (Pentoxifylline; PTX) on NF-κB and CREB activation in vitro in human mononuclear cells. With the use of specific inhibitors, we also investigated the role of PKA in LPS-induced TNF-α production. MATERIALS AND METHODS This study was approved by the Human Research Protections Program and the Institutional Review Board. Written consent to participate in the study was obtained from all volunteers prior to blood donation. Heparinized Vacutainers were purchased from Becton Dickinson (San Jose, CA).
T114 406-418 Sentence denotes INTRODUCTION
T6 406-418 Sentence denotes INTRODUCTION
T115 419-530 Sentence denotes The production of TNF-α following LPS stimulation is one of the key steps in bacterial sepsis and inflammation.
T7 419-530 Sentence denotes The production of TNF-α following LPS stimulation is one of the key steps in bacterial sepsis and inflammation.
T116 531-646 Sentence denotes The mechanism by which phosphodiesterase inhibition alters TNF-α production in the presence of LPS remains unclear.
T8 531-646 Sentence denotes The mechanism by which phosphodiesterase inhibition alters TNF-α production in the presence of LPS remains unclear.
T117 648-655 Sentence denotes METHODS
T9 648-655 Sentence denotes METHODS
T118 656-818 Sentence denotes Human mononuclear cells were stimulated with LPS (1 μg/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor.
T10 656-818 Sentence denotes Human mononuclear cells were stimulated with LPS (1 μg/mL), in the presence and absence of Pentoxifylline (PTX; 20 mM), a nonspecific phosphodiesterase inhibitor.
T119 819-975 Sentence denotes Western blotting of phosphorylated cytoplasmic I-κBα, nuclear factor-κB p65 (NF-κB), and nuclear cAMP-response element binding protein (CREB) was performed.
T11 819-975 Sentence denotes Western blotting of phosphorylated cytoplasmic I-κBα, nuclear factor-κB p65 (NF-κB), and nuclear cAMP-response element binding protein (CREB) was performed.
T120 976-1059 Sentence denotes DNA binding of NF-κB and CREB was verified by electrophoretic mobility shift assay.
T12 976-1059 Sentence denotes DNA binding of NF-κB and CREB was verified by electrophoretic mobility shift assay.
T121 1060-1232 Sentence denotes TNF-α levels were determined in the supernatant of stimulated cells in the presence and absence Protein kinase A inhibition by an enzyme-linked immunosorbent assay (ELISA).
T13 1060-1232 Sentence denotes TNF-α levels were determined in the supernatant of stimulated cells in the presence and absence Protein kinase A inhibition by an enzyme-linked immunosorbent assay (ELISA).
T122 1234-1241 Sentence denotes RESULTS
T14 1234-1241 Sentence denotes RESULTS
T123 1242-1389 Sentence denotes PTX was demonstrated to significantly reduce cytoplasmic I-κBα phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-κB.
T15 1242-1389 Sentence denotes PTX was demonstrated to significantly reduce cytoplasmic I-κBα phosphorylation, nuclear p65 phosphorylation, and the DNA binding activity of NF-κB.
T124 1390-1478 Sentence denotes In contrast, PTX markedly enhanced the phosphorylation and DNA binding activity of CREB.
T16 1390-1478 Sentence denotes In contrast, PTX markedly enhanced the phosphorylation and DNA binding activity of CREB.
T125 1479-1613 Sentence denotes Cells concomitantly treated with PTX and LPS secreted similar levels of TNF-α in the presence and absence Protein kinase A inhibition.
T17 1479-1613 Sentence denotes Cells concomitantly treated with PTX and LPS secreted similar levels of TNF-α in the presence and absence Protein kinase A inhibition.
T126 1615-1625 Sentence denotes DISCUSSION
T18 1615-1625 Sentence denotes DISCUSSION
T127 1626-1899 Sentence denotes The increased level of cAMP that results from phosphodiesterase inhibition affects cytoplasmic and nuclear events, resulting in the attenuation of NF-κB and the activation of CREB transcriptional DNA binding through pathways that are partially Protein kinase A-independent.
T19 1626-1899 Sentence denotes The increased level of cAMP that results from phosphodiesterase inhibition affects cytoplasmic and nuclear events, resulting in the attenuation of NF-κB and the activation of CREB transcriptional DNA binding through pathways that are partially Protein kinase A-independent.
T128 1901-1911 Sentence denotes CONCLUSION
T20 1901-1911 Sentence denotes CONCLUSION
T129 1912-2096 Sentence denotes PTX-mediated phosphodiesterase inhibition occurs partially through a Protein kinase A-independent pathway and may serve as a useful tool in the attenuation of LPS-induced inflammation.
T21 1912-2096 Sentence denotes PTX-mediated phosphodiesterase inhibition occurs partially through a Protein kinase A-independent pathway and may serve as a useful tool in the attenuation of LPS-induced inflammation.
T22 2099-2111 Sentence denotes INTRODUCTION
T1149 2112-2299 Sentence denotes The bacterial membrane component lipopolysaccharide (LPS) is capable of initiating phosphorylation and activation of multiple host intracellular protein kinases and transcription factors.
T23 2112-2299 Sentence denotes The bacterial membrane component lipopolysaccharide (LPS) is capable of initiating phosphorylation and activation of multiple host intracellular protein kinases and transcription factors.
T1150 2300-2486 Sentence denotes Transcription factor activation, which results in the modulation of gene transcription and protein synthesis, is a critical element in the defense mechanism of the host immune system.1,2
T24 2300-2486 Sentence denotes Transcription factor activation, which results in the modulation of gene transcription and protein synthesis, is a critical element in the defense mechanism of the host immune system.1,2
T1151 2487-2710 Sentence denotes LPS-induced transcription factor activation has been shown to be a key regulator of tumor necrosis factor-α (TNF-α) production.3 TNF-α is a potent pro-inflammatory cytokine involved in a wide spectrum of cellular responses.
T25 2487-2710 Sentence denotes LPS-induced transcription factor activation has been shown to be a key regulator of tumor necrosis factor-α (TNF-α) production.3 TNF-α is a potent pro-inflammatory cytokine involved in a wide spectrum of cellular responses.
T1152 2711-3055 Sentence denotes Furthermore, TNF-α synthesis can be attenuated in immune cells exposed to phosphodiesterase (PDE) inhibition after challenge by a variety of pro-inflammatory stimulants.4 The signaling mechanisms affected by PDE inhibition, which ultimately lead to the downregulation of TNF-α production, have not been well characterized in inflammatory cells.
T26 2711-3055 Sentence denotes Furthermore, TNF-α synthesis can be attenuated in immune cells exposed to phosphodiesterase (PDE) inhibition after challenge by a variety of pro-inflammatory stimulants.4 The signaling mechanisms affected by PDE inhibition, which ultimately lead to the downregulation of TNF-α production, have not been well characterized in inflammatory cells.
T1153 3056-3683 Sentence denotes Classically, it has been demonstrated that PDE inhibition results in the intracellular accumulation of the second messenger cyclic adenosine-3,5-monophosphate (cAMP) and subsequent activation of Protein kinase A (PKA).5 PKA activation then leads to the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), transmission of signals into the nucleus, and the subsequent modulation of gene transcription.6 This apparently simple linear cascade does not fully explain the mechanism by which an elevation in the intracellular cAMP level exerts wide-ranging effects on multiple cellular functions.
T27 3056-3683 Sentence denotes Classically, it has been demonstrated that PDE inhibition results in the intracellular accumulation of the second messenger cyclic adenosine-3,5-monophosphate (cAMP) and subsequent activation of Protein kinase A (PKA).5 PKA activation then leads to the phosphorylation of the transcription factor cAMP-response element binding protein (CREB), transmission of signals into the nucleus, and the subsequent modulation of gene transcription.6 This apparently simple linear cascade does not fully explain the mechanism by which an elevation in the intracellular cAMP level exerts wide-ranging effects on multiple cellular functions.
T1154 3684-3812 Sentence denotes There is a growing body of evidence suggesting that cAMP may function through both PKA-dependent and -independent mechanisms.6–8
T28 3684-3812 Sentence denotes There is a growing body of evidence suggesting that cAMP may function through both PKA-dependent and -independent mechanisms.6–8
T1155 3813-3942 Sentence denotes LPS-induced activation of the transcription factor nuclear factor-κB (NF-κB) has also been the focus of a great deal of research.
T29 3813-3942 Sentence denotes LPS-induced activation of the transcription factor nuclear factor-κB (NF-κB) has also been the focus of a great deal of research.
T1156 3943-4119 Sentence denotes It has been clearly demonstrated that agents that increase intracellular cAMP also inhibit NF-κB-dependent pro-inflammatory gene transcription, particularly of the TNF-α gene.9
T30 3943-4119 Sentence denotes It has been clearly demonstrated that agents that increase intracellular cAMP also inhibit NF-κB-dependent pro-inflammatory gene transcription, particularly of the TNF-α gene.9
T1157 4120-4228 Sentence denotes Controversy exists regarding the exact mechanism(s) by which PDE inhibition down-regulates TNF-α production.
T31 4120-4228 Sentence denotes Controversy exists regarding the exact mechanism(s) by which PDE inhibition down-regulates TNF-α production.
T1158 4229-4494 Sentence denotes Possibilities include, but are not limited to, inhibition of NF-κB DNA binding activity, downregulation of NF-κB transcriptional activity, increased CREB activation, and competition between NF-κB and CREB for common co-activators such as CREB binding protein (CBP).
T32 4229-4494 Sentence denotes Possibilities include, but are not limited to, inhibition of NF-κB DNA binding activity, downregulation of NF-κB transcriptional activity, increased CREB activation, and competition between NF-κB and CREB for common co-activators such as CREB binding protein (CBP).
T1159 4495-4579 Sentence denotes It is also not known whether these processes rely solely upon the activation of PKA.
T33 4495-4579 Sentence denotes It is also not known whether these processes rely solely upon the activation of PKA.
T1160 4580-4812 Sentence denotes Therefore, the objective of the present study is to determine the effects of nonspecific PDE inhibition with 1-[5-oxohexyl]-3,7-dimethylxanthine (Pentoxifylline; PTX) on NF-κB and CREB activation in vitro in human mononuclear cells.
T34 4580-4812 Sentence denotes Therefore, the objective of the present study is to determine the effects of nonspecific PDE inhibition with 1-[5-oxohexyl]-3,7-dimethylxanthine (Pentoxifylline; PTX) on NF-κB and CREB activation in vitro in human mononuclear cells.
T1161 4813-4919 Sentence denotes With the use of specific inhibitors, we also investigated the role of PKA in LPS-induced TNF-α production.
T35 4813-4919 Sentence denotes With the use of specific inhibitors, we also investigated the role of PKA in LPS-induced TNF-α production.
T36 4921-4942 Sentence denotes MATERIALS AND METHODS
T37 4943-5044 Sentence denotes This study was approved by the Human Research Protections Program and the Institutional Review Board.
T38 5045-5146 Sentence denotes Written consent to participate in the study was obtained from all volunteers prior to blood donation.
T39 5147-5223 Sentence denotes Heparinized Vacutainers were purchased from Becton Dickinson (San Jose, CA).
T2454 5224-5312 Sentence denotes 1.5-mL Eppendorf centrifuge tubes were purchased from Fisher Scientific (Pittsburg, PA).
T40 5224-5312 Sentence denotes 1.5-mL Eppendorf centrifuge tubes were purchased from Fisher Scientific (Pittsburg, PA).
T2455 5313-5417 Sentence denotes Hank’s balanced salt solution (HBSS) and RPMI 1640 were obtained from Irvine Scientific (Santa Ana, CA).
T41 5313-5417 Sentence denotes Hank’s balanced salt solution (HBSS) and RPMI 1640 were obtained from Irvine Scientific (Santa Ana, CA).
T2456 5418-5511 Sentence denotes LPS from Escherichia coli serotype 0111:B4 and PTX were purchased from Sigma (St. Louis, MO).
T42 5418-5511 Sentence denotes LPS from Escherichia coli serotype 0111:B4 and PTX were purchased from Sigma (St. Louis, MO).
T2457 5512-5651 Sentence denotes The PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoguinolinesulfonamide-2HCl] (H89), was purchased from Calbiochem (La Jolla, CA).
T43 5512-5651 Sentence denotes The PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoguinolinesulfonamide-2HCl] (H89), was purchased from Calbiochem (La Jolla, CA).
T44 5652-5864 Sentence denotes Antibodies for phosphorylated CREB (serine 133), phosphorylated I-κBα (serines 32 and 36), phosphorylated NF-κB p65 subunit (serine 276), and secondary antibodies were purchased from Cell Signaling (Beverly, MA).
T2458 5652-5961 Sentence denotes Antibodies for phosphorylated CREB (serine 133), phosphorylated I-κBα (serines 32 and 36), phosphorylated NF-κB p65 subunit (serine 276), and secondary antibodies were purchased from Cell Signaling (Beverly, MA). Dextran T500 and Percoll were received from GE Healthcare/Amersham Biosciences (Piscataway, NJ).
T45 5865-5961 Sentence denotes Dextran T500 and Percoll were received from GE Healthcare/Amersham Biosciences (Piscataway, NJ).
T46 5962-6077 Sentence denotes The enzyme-linked immunosorbent assay (ELISA) for TNF-α was obtained from Quantikine R&D Systems (Minneapolis, MN).
T2459 5962-6221 Sentence denotes The enzyme-linked immunosorbent assay (ELISA) for TNF-α was obtained from Quantikine R&D Systems (Minneapolis, MN). Tris-glycine and DNA retardation gels, nitrocellulose membranes, and western blot running buffers were obtained from Invitrogen (Carlsbad, CA).
T47 6078-6221 Sentence denotes Tris-glycine and DNA retardation gels, nitrocellulose membranes, and western blot running buffers were obtained from Invitrogen (Carlsbad, CA).
T2460 6222-6439 Sentence denotes NE-PER® nuclear and cytoplasmic extraction reagents, the BCA protein reagent kit, the Supersignal West Pico Chemiluminescent Kit, and the LightShift Chemiluminescent EMSA Kit were purchased from Pierce (Rockland, IL).
T48 6222-6439 Sentence denotes NE-PER® nuclear and cytoplasmic extraction reagents, the BCA protein reagent kit, the Supersignal West Pico Chemiluminescent Kit, and the LightShift Chemiluminescent EMSA Kit were purchased from Pierce (Rockland, IL).
T2461 6440-6523 Sentence denotes CREB and NF-κB DNA oligonucleotide probes were synthesized by IDT (Coralville, IA).
T49 6440-6523 Sentence denotes CREB and NF-κB DNA oligonucleotide probes were synthesized by IDT (Coralville, IA).
T2462 6524-6602 Sentence denotes Nylon membranes were purchased from Roche Applied Sciences (Indianapolis, IN).
T50 6524-6602 Sentence denotes Nylon membranes were purchased from Roche Applied Sciences (Indianapolis, IN).
T2463 6603-6722 Sentence denotes Western blot band quantification was performed with the UN-SCAN-IT Gel Digitizing software (Silk Scientific, Orem, UT).
T51 6603-6722 Sentence denotes Western blot band quantification was performed with the UN-SCAN-IT Gel Digitizing software (Silk Scientific, Orem, UT).
T3255 6724-6789 Sentence denotes Human Peripheral Blood Mononuclear Cell Isolation and Stimulation
T52 6724-6789 Sentence denotes Human Peripheral Blood Mononuclear Cell Isolation and Stimulation
T3256 6790-6887 Sentence denotes Human mononuclear cells were isolated from the peripheral blood of four healthy human volunteers.
T53 6790-6887 Sentence denotes Human mononuclear cells were isolated from the peripheral blood of four healthy human volunteers.
T3257 6888-7093 Sentence denotes The sample size was chosen from our previously published work, which demonstrated reliable results.10 Cell isolation and all subsequent experiments were conducted under sterile and pyrogen-free conditions.
T54 6888-7093 Sentence denotes The sample size was chosen from our previously published work, which demonstrated reliable results.10 Cell isolation and all subsequent experiments were conducted under sterile and pyrogen-free conditions.
T3258 7094-7228 Sentence denotes Blood collected in heparin tubes was incubated with Dextran T500 and the red cells were sedimented for 40 minutes at room temperature.
T55 7094-7228 Sentence denotes Blood collected in heparin tubes was incubated with Dextran T500 and the red cells were sedimented for 40 minutes at room temperature.
T3259 7229-7369 Sentence denotes The resultant serum was then washed with HBSS and separated by Percoll gradient centrifugation according to the manufacturer’s instructions.
T56 7229-7369 Sentence denotes The resultant serum was then washed with HBSS and separated by Percoll gradient centrifugation according to the manufacturer’s instructions.
T3260 7370-7460 Sentence denotes Cell viability was assessed by trypan blue dye exclusion, with purity of greater than 95%.
T57 7370-7460 Sentence denotes Cell viability was assessed by trypan blue dye exclusion, with purity of greater than 95%.
T3261 7461-7585 Sentence denotes Isolated cells were resuspended in RPMI 1640 supplemented with 10% FBS and 5 mM HEPES at a concentration of 1 x 107cells/mL.
T58 7461-7585 Sentence denotes Isolated cells were resuspended in RPMI 1640 supplemented with 10% FBS and 5 mM HEPES at a concentration of 1 x 107cells/mL.
T3262 7586-7688 Sentence denotes Each subsequent experiment listed below was conducted on samples of 5 x 106 cells per treatment group.
T59 7586-7688 Sentence denotes Each subsequent experiment listed below was conducted on samples of 5 x 106 cells per treatment group.
T3263 7689-7876 Sentence denotes Isolated cells were stimulated with either HBSS as a negative control, LPS (1 μg/mL), PTX (20 mM), or concomitant LPS / PTX, at the above-mentioned concentrations, for 30 minutes at 37ºC.
T60 7689-7876 Sentence denotes Isolated cells were stimulated with either HBSS as a negative control, LPS (1 μg/mL), PTX (20 mM), or concomitant LPS / PTX, at the above-mentioned concentrations, for 30 minutes at 37ºC.
T3264 7877-8001 Sentence denotes Cells were placed on ice for 10 minutes to stop the reaction and the samples were then stored at −70°C for further analysis.
T61 7877-8001 Sentence denotes Cells were placed on ice for 10 minutes to stop the reaction and the samples were then stored at −70°C for further analysis.
T3265 8002-8242 Sentence denotes The stimulation times and concentrations of LPS and PTX utilized in this study were determined in previous pilot studies done in our laboratory, which examined the effects of increasing concentrations of PTX on LPS-induced TNF-α production.
T62 8002-8242 Sentence denotes The stimulation times and concentrations of LPS and PTX utilized in this study were determined in previous pilot studies done in our laboratory, which examined the effects of increasing concentrations of PTX on LPS-induced TNF-α production.
T3266 8243-8503 Sentence denotes LPS at a concentration of 1 μg/mL and PTX at a concentration of 20 mM were the minimum concentrations necessary to produce a reproducible and reliable up-regulation and down-regulation in TNF-α production, respectively, in quantitative assays (data not shown).
T63 8243-8503 Sentence denotes LPS at a concentration of 1 μg/mL and PTX at a concentration of 20 mM were the minimum concentrations necessary to produce a reproducible and reliable up-regulation and down-regulation in TNF-α production, respectively, in quantitative assays (data not shown).
T3968 8505-8571 Sentence denotes Effect of PTX on LPS-induced TNF-α production by mononuclear cells
T64 8505-8571 Sentence denotes Effect of PTX on LPS-induced TNF-α production by mononuclear cells
T3969 8572-8773 Sentence denotes The concentration of TNF-α was quantitatively determined by ELISA in the media of cells exposed to the treatments described above after stimulation for up to a maximum of 18 hours, at 3-hour intervals.
T65 8572-8773 Sentence denotes The concentration of TNF-α was quantitatively determined by ELISA in the media of cells exposed to the treatments described above after stimulation for up to a maximum of 18 hours, at 3-hour intervals.
T3970 8774-8809 Sentence denotes The results are expressed in pg/mL.
T66 8774-8809 Sentence denotes The results are expressed in pg/mL.
T4138 8811-8867 Sentence denotes SDS-PAGE and Immunoblotting (I-κBα, NF-κB p65, and CREB)
T67 8811-8867 Sentence denotes SDS-PAGE and Immunoblotting (I-κBα, NF-κB p65, and CREB)
T4139 8868-8968 Sentence denotes Following stimulation, PBMCs were washed with ice-cold PBS and centrifuged to collect a cell pellet.
T68 8868-8968 Sentence denotes Following stimulation, PBMCs were washed with ice-cold PBS and centrifuged to collect a cell pellet.
T4140 8969-9066 Sentence denotes The pellet was resuspended in ice-cold SDS sample buffer supplemented with 100 mM dithiothreitol.
T69 8969-9066 Sentence denotes The pellet was resuspended in ice-cold SDS sample buffer supplemented with 100 mM dithiothreitol.
T4141 9067-9256 Sentence denotes Cytoplasmic and nuclear extracts were isolated with NE-PER nuclear and cytoplasmic extraction reagents with 1x Halt Protease Inhibitor Cocktail according to the manufacturer’s instructions.
T70 9067-9256 Sentence denotes Cytoplasmic and nuclear extracts were isolated with NE-PER nuclear and cytoplasmic extraction reagents with 1x Halt Protease Inhibitor Cocktail according to the manufacturer’s instructions.
T4142 9257-9391 Sentence denotes Protein concentrations were determined with the BCA protein reagent kit for each sample according to a standardized curve for albumin.
T71 9257-9391 Sentence denotes Protein concentrations were determined with the BCA protein reagent kit for each sample according to a standardized curve for albumin.
T4143 9392-9479 Sentence denotes Cell lysates were collected by boiling the samples in a 100ºC water bath for 5 minutes.
T72 9392-9479 Sentence denotes Cell lysates were collected by boiling the samples in a 100ºC water bath for 5 minutes.
T4144 9480-9665 Sentence denotes Ten μg of protein per sample was separated by SDS-polyacrylamide gel electrophoresis through 8–16% tris-glycine polyacrylamide gradient gels and transferred to nitrocellulose membranes.
T73 9480-9665 Sentence denotes Ten μg of protein per sample was separated by SDS-polyacrylamide gel electrophoresis through 8–16% tris-glycine polyacrylamide gradient gels and transferred to nitrocellulose membranes.
T4145 9666-9787 Sentence denotes The membranes were blocked with 5% milk in Tris-buffered saline/Tween 20 (Fischer Scientific, Pittsburgh, PA) for 1 hour.
T74 9666-9787 Sentence denotes The membranes were blocked with 5% milk in Tris-buffered saline/Tween 20 (Fischer Scientific, Pittsburgh, PA) for 1 hour.
T4146 9788-10041 Sentence denotes Cytoplasmic extracts were incubated with phosphorylated I-κBα antibody (1:200), while nuclear extracts were incubated with either phosphorylated NF-κB p65 antibody (1:500) or phosphorylated CREB antibody (1:500) overnight at 4°C in separate experiments.
T75 9788-10041 Sentence denotes Cytoplasmic extracts were incubated with phosphorylated I-κBα antibody (1:200), while nuclear extracts were incubated with either phosphorylated NF-κB p65 antibody (1:500) or phosphorylated CREB antibody (1:500) overnight at 4°C in separate experiments.
T4147 10042-10259 Sentence denotes The membranes were washed with Tris-buffered saline/Tween 20 and incubated for 1 hour at room temperature with the secondary antibody, horseradish peroxidase-linked anti-rabbit IgG diluted 1:2000 in blocking solution.
T76 10042-10259 Sentence denotes The membranes were washed with Tris-buffered saline/Tween 20 and incubated for 1 hour at room temperature with the secondary antibody, horseradish peroxidase-linked anti-rabbit IgG diluted 1:2000 in blocking solution.
T4148 10260-10418 Sentence denotes After repeated washing of the membrane, the Supersignal West Pico Chemiluminescent Kit was applied for antibody detection per the manufacturer’s instructions.
T77 10260-10418 Sentence denotes After repeated washing of the membrane, the Supersignal West Pico Chemiluminescent Kit was applied for antibody detection per the manufacturer’s instructions.
T4149 10419-10485 Sentence denotes Western blot data is presented as a percentage of LPS stimulation.
T78 10419-10485 Sentence denotes Western blot data is presented as a percentage of LPS stimulation.
T4150 10486-10682 Sentence denotes The percentage of LPS stimulation was calculated by dividing the mean band pixel total for each treatment arm divided by the mean pixel total of samples stimulated with LPS and multiplying by 100.
T79 10486-10682 Sentence denotes The percentage of LPS stimulation was calculated by dividing the mean band pixel total for each treatment arm divided by the mean pixel total of samples stimulated with LPS and multiplying by 100.
T4151 10683-10800 Sentence denotes Thus, LPS stimulation is reported as 100% and each of the treatment arms is reported as a percent of LPS stimulation.
T80 10683-10800 Sentence denotes Thus, LPS stimulation is reported as 100% and each of the treatment arms is reported as a percent of LPS stimulation.
T4895 10802-10860 Sentence denotes NF-κB and CREB Electrophoretic Mobility Shift Assay (EMSA)
T81 10802-10860 Sentence denotes NF-κB and CREB Electrophoretic Mobility Shift Assay (EMSA)
T4896 10861-10962 Sentence denotes The non-radioactive LightShift Chemiluminescent EMSA Kit was used to detect DNA-protein interactions.
T82 10861-10962 Sentence denotes The non-radioactive LightShift Chemiluminescent EMSA Kit was used to detect DNA-protein interactions.
T4897 10963-11202 Sentence denotes The NF-κB 3′ biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 42-bp double stranded construct (5′-TTGTTACAA-GGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC – 3′) containing two tandemly repeated NF-κB binding sites (underlined).
T83 10963-11202 Sentence denotes The NF-κB 3′ biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 42-bp double stranded construct (5′-TTGTTACAA-GGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC – 3′) containing two tandemly repeated NF-κB binding sites (underlined).
T4898 11203-11342 Sentence denotes Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded NF-κB oligonucleotide.
T84 11203-11342 Sentence denotes Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded NF-κB oligonucleotide.
T4899 11343-11550 Sentence denotes The CREB 3′ biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 23-bp double stranded construct (5′-TTT TCG AGC TCTGACGTCAGA-GC – 3′) containing the CRE consensus sequence (underlined).
T85 11343-11550 Sentence denotes The CREB 3′ biotin end-labeled DNA oligonucleotide used as a probe for the EMSA was a 23-bp double stranded construct (5′-TTT TCG AGC TCTGACGTCAGA-GC – 3′) containing the CRE consensus sequence (underlined).
T4900 11551-11689 Sentence denotes Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded CREB oligonucleotide.
T86 11551-11689 Sentence denotes Specificity was determined by a competition assay with the addition of 200 molar excess of unlabeled double stranded CREB oligonucleotide.
T4901 11690-11767 Sentence denotes Nuclear extracts (10 μg) were incubated with 5 nM NF-κB or CREB probe (NF-kB:
T87 11690-11767 Sentence denotes Nuclear extracts (10 μg) were incubated with 5 nM NF-κB or CREB probe (NF-kB:
T4902 11768-11874 Sentence denotes 1x binding buffer, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1% NP40, 10% glycerol, and 50 ng/μl poly dI-dC, CREB:
T88 11768-11874 Sentence denotes 1x binding buffer, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.1% NP40, 10% glycerol, and 50 ng/μl poly dI-dC, CREB:
T4903 11875-12096 Sentence denotes 1x binding buffer, 20 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.10% NP40, 6% glycerol, 0.1 mg/mL BSA and 50 ng/μl poly dI-dC) and were then electrophoresed through a 6% DNA retardation gel at 100V for 90 minutes.
T89 11875-12096 Sentence denotes 1x binding buffer, 20 mM Tris, pH 7.5, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 0.10% NP40, 6% glycerol, 0.1 mg/mL BSA and 50 ng/μl poly dI-dC) and were then electrophoresed through a 6% DNA retardation gel at 100V for 90 minutes.
T4904 12097-12308 Sentence denotes The gels were electrophoretically transferred at 380mA for 1 hour on ice to a positively charged nylon membrane and immediately cross-linked for 15 minutes with a UV transilluminator equipped with a 312 nm bulb.
T90 12097-12308 Sentence denotes The gels were electrophoretically transferred at 380mA for 1 hour on ice to a positively charged nylon membrane and immediately cross-linked for 15 minutes with a UV transilluminator equipped with a 312 nm bulb.
T4905 12309-12448 Sentence denotes Streptavidin-horseradish peroxidase conjugate and the LightShift Chemiluminescent Substrate were used to detect the biotin end-labeled DNA.
T91 12309-12448 Sentence denotes Streptavidin-horseradish peroxidase conjugate and the LightShift Chemiluminescent Substrate were used to detect the biotin end-labeled DNA.
T4906 12449-12526 Sentence denotes The nylon membranes were exposed to x-ray film for 1–3 minutes for detection.
T92 12449-12526 Sentence denotes The nylon membranes were exposed to x-ray film for 1–3 minutes for detection.
T5643 12528-12588 Sentence denotes Role of PKA on LPS-induced mononuclear cell TNF-α production
T93 12528-12588 Sentence denotes Role of PKA on LPS-induced mononuclear cell TNF-α production
T5644 12589-12807 Sentence denotes To evaluate the role of PKA on TNF-α production in mononuclear cells and its involvement in the attenuation of LPS-induced TNF-α production observed following PTX treatment, H89, a specific PKA inhibitor, was utilized.
T94 12589-12807 Sentence denotes To evaluate the role of PKA on TNF-α production in mononuclear cells and its involvement in the attenuation of LPS-induced TNF-α production observed following PTX treatment, H89, a specific PKA inhibitor, was utilized.
T5645 12808-12980 Sentence denotes Isolated mononuclear cells were incubated according to the treatment groups described above in the presence and absence of pretreatment with H89 (10 μM) for 1 hour at 37ºC.
T95 12808-12980 Sentence denotes Isolated mononuclear cells were incubated according to the treatment groups described above in the presence and absence of pretreatment with H89 (10 μM) for 1 hour at 37ºC.
T5646 12981-13299 Sentence denotes The dose of H89 was chosen based on previous work which demonstrated specific and complete inhibition of PKA at this concentration.10 Since the activity of this inhibitor is both specific and consistent at this specified dose, we did not include experiments with additional PKA inhibitors with this set of experiments.
T96 12981-13299 Sentence denotes The dose of H89 was chosen based on previous work which demonstrated specific and complete inhibition of PKA at this concentration.10 Since the activity of this inhibitor is both specific and consistent at this specified dose, we did not include experiments with additional PKA inhibitors with this set of experiments.
T5647 13300-13380 Sentence denotes The TNF-α concentration in the supernatant was measured quantitatively by ELISA.
T97 13300-13380 Sentence denotes The TNF-α concentration in the supernatant was measured quantitatively by ELISA.
T5648 13381-13416 Sentence denotes The results are expressed in pg/mL.
T98 13381-13416 Sentence denotes The results are expressed in pg/mL.
T6066 13418-13438 Sentence denotes Statistical Analysis
T99 13418-13438 Sentence denotes Statistical Analysis
T6067 13439-13561 Sentence denotes The experimental results obtained in this study were derived from four separate experiments with healthy volunteer donors.
T100 13439-13561 Sentence denotes The experimental results obtained in this study were derived from four separate experiments with healthy volunteer donors.
T6068 13562-13632 Sentence denotes Each assay was performed in duplicate or triplicate where appropriate.
T101 13562-13632 Sentence denotes Each assay was performed in duplicate or triplicate where appropriate.
T6069 13633-13669 Sentence denotes Data is presented as the mean ± SEM.
T102 13633-13669 Sentence denotes Data is presented as the mean ± SEM.
T6070 13670-13794 Sentence denotes Statistical differences between groups were determined by one-way analysis of variance (ANOVA) with a Bonferroni correction.
T103 13670-13794 Sentence denotes Statistical differences between groups were determined by one-way analysis of variance (ANOVA) with a Bonferroni correction.
T6071 13795-13844 Sentence denotes Statistical significance was defined as P < 0.05.
T104 13795-13844 Sentence denotes Statistical significance was defined as P < 0.05.
T105 13846-13853 Sentence denotes RESULTS
T6239 13855-13905 Sentence denotes The effects of PTX on LPS-induced TNF-α production
T106 13855-13905 Sentence denotes The effects of PTX on LPS-induced TNF-α production
T6240 13906-14028 Sentence denotes To determine the duration of PDE inhibition on TNF-α production after LPS stimulation, time course studies were conducted.
T107 13906-14028 Sentence denotes To determine the duration of PDE inhibition on TNF-α production after LPS stimulation, time course studies were conducted.
T6241 14029-14137 Sentence denotes In accordance with prior work, PTX had a rapid and sustained effect on TNF-α production in vitro (Figure 1).
T108 14029-14137 Sentence denotes In accordance with prior work, PTX had a rapid and sustained effect on TNF-α production in vitro (Figure 1).
T6497 14139-14181 Sentence denotes PTX decreases LPS-induced NF-κB activation
T109 14139-14181 Sentence denotes PTX decreases LPS-induced NF-κB activation
T6498 14182-14346 Sentence denotes Since phosphorylation leads to ubiquitination and degradation of I-κBα and subsequent nuclear translocation of NF-κB, we first examined the effects of PTX on I-κBα.
T110 14182-14346 Sentence denotes Since phosphorylation leads to ubiquitination and degradation of I-κBα and subsequent nuclear translocation of NF-κB, we first examined the effects of PTX on I-κBα.
T6499 14347-14487 Sentence denotes Cytoplasmic I-κBα phosphorylation was markedly increased following LPS stimulation when compared to control (100 ± 0 vs. 20 ± 18; P < 0.05).
T111 14347-14487 Sentence denotes Cytoplasmic I-κBα phosphorylation was markedly increased following LPS stimulation when compared to control (100 ± 0 vs. 20 ± 18; P < 0.05).
T6500 14488-14590 Sentence denotes The addition of PTX significantly downregulated LPS-induced I-κBα phosphorylation (P = 0.02; Figure 2)
T112 14488-14590 Sentence denotes The addition of PTX significantly downregulated LPS-induced I-κBα phosphorylation (P = 0.02; Figure 2)
T6501 14591-14719 Sentence denotes In a similar fashion, nuclear NF-κB p65 phosphorylation was increased following LPS stimulation (100 ± 0 vs. 38 ± 20; P < 0.01).
T113 14591-14719 Sentence denotes In a similar fashion, nuclear NF-κB p65 phosphorylation was increased following LPS stimulation (100 ± 0 vs. 38 ± 20; P < 0.01).
T6502 14720-14887 Sentence denotes PTX similarly decreased LPS-induced NF-κB p65 nuclear phosphorylation, a marker of NF-κB activation and nuclear translocation (100 ± 0 vs. 40 ± 6; P =0.03; Figure 3A).
T114 14720-14887 Sentence denotes PTX similarly decreased LPS-induced NF-κB p65 nuclear phosphorylation, a marker of NF-κB activation and nuclear translocation (100 ± 0 vs. 40 ± 6; P =0.03; Figure 3A).
T6503 14888-15044 Sentence denotes EMSAs were then performed to verify the PTX-induced alterations in nuclear phosphorylation resulted in similar effects on the DNA binding activity of NF-κB.
T115 14888-15044 Sentence denotes EMSAs were then performed to verify the PTX-induced alterations in nuclear phosphorylation resulted in similar effects on the DNA binding activity of NF-κB.
T6504 15045-15240 Sentence denotes The addition of PTX to LPS-stimulated mononuclear cells resulted in comparable downregulation of DNA binding activity, similar to the observed downregulation of NF-κB phosphorylation (Figure 3B).
T116 15045-15240 Sentence denotes The addition of PTX to LPS-stimulated mononuclear cells resulted in comparable downregulation of DNA binding activity, similar to the observed downregulation of NF-κB phosphorylation (Figure 3B).
T7083 15242-15315 Sentence denotes PTX upregulates CREB phosphorylation and activation after LPS stimulation
T117 15242-15315 Sentence denotes PTX upregulates CREB phosphorylation and activation after LPS stimulation
T7084 15316-15389 Sentence denotes Phosphorylation of nuclear CREB was used as a marker for CREB activation.
T118 15316-15389 Sentence denotes Phosphorylation of nuclear CREB was used as a marker for CREB activation.
T7085 15390-15496 Sentence denotes LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A).
T119 15390-15496 Sentence denotes LPS stimulation caused a negligible increase in CREB phosphorylation when compared to control (Figure 4A).
T7086 15497-15576 Sentence denotes PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS).
T120 15497-15576 Sentence denotes PTX alone caused a marked increase in CREB phosphorylation (P < 0.01 vs. HBSS).
T7087 15577-15739 Sentence denotes When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 ± 92 vs. 100 ± 0; P < 0.01).
T121 15577-15739 Sentence denotes When LPS and PTX exposure occurred simultaneously, CREB phosphorylation was significantly higher than with LPS stimulation alone (543 ± 92 vs. 100 ± 0; P < 0.01).
T7088 15740-15942 Sentence denotes In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).
T122 15740-15942 Sentence denotes In addition, the amount of CREB phosphorylation seen with concomitant LPS and PTX treatment was less than that seen with PTX alone, although this difference was not statistically significant (P = 0.08).
T7089 15943-16063 Sentence denotes To determine if CREB-DNA binding was affected by PTX in a manner similar to CREB phosphorylation, an EMSA was performed.
T123 15943-16063 Sentence denotes To determine if CREB-DNA binding was affected by PTX in a manner similar to CREB phosphorylation, an EMSA was performed.
T7090 16064-16175 Sentence denotes Exposure of LPS-stimulated cells to PTX similarly increased CREB DNA-binding compared to LPS alone (Figure 4B).
T124 16064-16175 Sentence denotes Exposure of LPS-stimulated cells to PTX similarly increased CREB DNA-binding compared to LPS alone (Figure 4B).
T7606 16177-16244 Sentence denotes Effect of PTX on TNF-α production in the presence of PKA inhibition
T125 16177-16244 Sentence denotes Effect of PTX on TNF-α production in the presence of PKA inhibition
T7607 16245-16447 Sentence denotes The role of PKA in PTX-induced downregulation of TNF-α production in LPS-stimulated mononuclear cells was assessed by treating mononuclear cells with H89, a PKA inhibitor, prior to LPS and PTX exposure.
T126 16245-16447 Sentence denotes The role of PKA in PTX-induced downregulation of TNF-α production in LPS-stimulated mononuclear cells was assessed by treating mononuclear cells with H89, a PKA inhibitor, prior to LPS and PTX exposure.
T7608 16448-16622 Sentence denotes The addition PTX to LPS-stimulated cells resulted in a reduction in TNF-α, similar to that observed in our previous experiments (529 pg/mL ± 112 vs. 37 pg/mL ± 10; P < 0.01).
T127 16448-16622 Sentence denotes The addition PTX to LPS-stimulated cells resulted in a reduction in TNF-α, similar to that observed in our previous experiments (529 pg/mL ± 112 vs. 37 pg/mL ± 10; P < 0.01).
T7609 16623-16821 Sentence denotes Pre-incubation of cells with H89 prior to LPS and PTX exposure did not result in any significant modulation of TNF-α production when compared to that demonstrated with PTX alone (P = 0.2; Figure 5).
T128 16623-16821 Sentence denotes Pre-incubation of cells with H89 prior to LPS and PTX exposure did not result in any significant modulation of TNF-α production when compared to that demonstrated with PTX alone (P = 0.2; Figure 5).
T7610 16822-16965 Sentence denotes These results suggest that downregulation of TNF-α production by PTX in LPS-stimulated mononuclear cells is, at least in part, PKA-independent.
T129 16822-16965 Sentence denotes These results suggest that downregulation of TNF-α production by PTX in LPS-stimulated mononuclear cells is, at least in part, PKA-independent.
T130 16967-16977 Sentence denotes DISCUSSION
T8375 16978-17564 Sentence denotes The exposure of inflammatory cells to a variety of extracellular stimuli results in the initiation of transcriptional and posttranscriptional events, which culminate in the production of pro-inflammatory mediators.11–14 TNF-α is released very early following shock and is considered an important mediator of the inflammatory cascade, since it regulates the synthesis of several other critical cytokines and chemokines.15 A clear relationship between increased TNF-α synthesis and the development of shock and multiple organ dysfunction has been observed in animal models of sepsis.15–17
T131 16978-17564 Sentence denotes The exposure of inflammatory cells to a variety of extracellular stimuli results in the initiation of transcriptional and posttranscriptional events, which culminate in the production of pro-inflammatory mediators.11–14 TNF-α is released very early following shock and is considered an important mediator of the inflammatory cascade, since it regulates the synthesis of several other critical cytokines and chemokines.15 A clear relationship between increased TNF-α synthesis and the development of shock and multiple organ dysfunction has been observed in animal models of sepsis.15–17
T8376 17565-17743 Sentence denotes Because of its importance in orchestrating multiple steps of the inflammatory response, strategies aimed at decreasing TNF-α levels or attenuating its production seem attractive.
T132 17565-17743 Sentence denotes Because of its importance in orchestrating multiple steps of the inflammatory response, strategies aimed at decreasing TNF-α levels or attenuating its production seem attractive.
T8377 17744-17843 Sentence denotes PDE inhibitors have been shown to downregulate TNF-α production in distinct human cell populations.
T133 17744-17843 Sentence denotes PDE inhibitors have been shown to downregulate TNF-α production in distinct human cell populations.
T8378 17844-18196 Sentence denotes We have previously shown that, in general, TNF-α levels continue to increase after LPS exposure and that concomitant infusion of phosphodiesterase inhibitors can markedly decrease TNF-α levels.18–19 In this in vitro study, we verified the sustained attenuation of LPS-induced TNF-α expression over time in an isolated human mononuclear cell population.
T134 17844-18196 Sentence denotes We have previously shown that, in general, TNF-α levels continue to increase after LPS exposure and that concomitant infusion of phosphodiesterase inhibitors can markedly decrease TNF-α levels.18–19 In this in vitro study, we verified the sustained attenuation of LPS-induced TNF-α expression over time in an isolated human mononuclear cell population.
T8379 18197-18364 Sentence denotes The mechanisms by which PTX, a non-specific phosphodiesterase inhibitor, significantly and consistently decreases TNF-α production have not been completely elucidated.
T135 18197-18364 Sentence denotes The mechanisms by which PTX, a non-specific phosphodiesterase inhibitor, significantly and consistently decreases TNF-α production have not been completely elucidated.
T8380 18365-18500 Sentence denotes It has been postulated that drugs that increase intracellular cAMP exert their anti-inflammatory effects through the activation of PKA.
T136 18365-18500 Sentence denotes It has been postulated that drugs that increase intracellular cAMP exert their anti-inflammatory effects through the activation of PKA.
T8381 18501-18908 Sentence denotes This classic pathway has been challenged as the sole mechanism involved in the modulation of inflammation by cAMP-enhancing drugs, and alternative mechanisms involving PKA-independent pathways have been proposed.6–8 In this series of experiments, we observed a significant attenuation of TNF-α production in LPS-stimulated human mononuclear cells following PTX administration, independent of PKA activation.
T137 18501-18908 Sentence denotes This classic pathway has been challenged as the sole mechanism involved in the modulation of inflammation by cAMP-enhancing drugs, and alternative mechanisms involving PKA-independent pathways have been proposed.6–8 In this series of experiments, we observed a significant attenuation of TNF-α production in LPS-stimulated human mononuclear cells following PTX administration, independent of PKA activation.
T8382 18909-19332 Sentence denotes We used a nonspecific phosphodiesterase inhibitor, PTX, in the present study because it has been used clinically in the treatment of a variety of conditions in which inflammation is an important component of the pathophysiology of the disease process.19 Additionally, the use of PTX in sepsis as an adjunct to other treatments to maintain adequate organ function has been explored based upon its effects on TNF-α synthesis.
T138 18909-19332 Sentence denotes We used a nonspecific phosphodiesterase inhibitor, PTX, in the present study because it has been used clinically in the treatment of a variety of conditions in which inflammation is an important component of the pathophysiology of the disease process.19 Additionally, the use of PTX in sepsis as an adjunct to other treatments to maintain adequate organ function has been explored based upon its effects on TNF-α synthesis.
T8383 19333-19834 Sentence denotes However, the reported beneficial effects of PTX are not only related to the downregulation of TNF-αbut also to PTX’s hemorrheologic properties, its ability to reduce neutrophil activation, and its beneficial effects on microcirculation, cardiac performance, and organ injury.20–25 Herein, we demonstrated that the downregulatory effects of PTX in human mononuclear cells involves modulation of pathways that involve NF-κB and CREB, two major transcription factors involved in the inflammatory cascade.
T139 19333-19834 Sentence denotes However, the reported beneficial effects of PTX are not only related to the downregulation of TNF-αbut also to PTX’s hemorrheologic properties, its ability to reduce neutrophil activation, and its beneficial effects on microcirculation, cardiac performance, and organ injury.20–25 Herein, we demonstrated that the downregulatory effects of PTX in human mononuclear cells involves modulation of pathways that involve NF-κB and CREB, two major transcription factors involved in the inflammatory cascade.
T8384 19835-19998 Sentence denotes NF-κB/Rel constitutes a family of transcriptional factors involved in the regulation of numerous cytokine genes and immune responses in different cell populations.
T140 19835-19998 Sentence denotes NF-κB/Rel constitutes a family of transcriptional factors involved in the regulation of numerous cytokine genes and immune responses in different cell populations.
T8385 19999-20160 Sentence denotes The most abundant form of NF-κB is the p50–p65 heterodimer.26 The inactive form of NF-κB exists in the cytoplasm bound to an inhibitory complex containing I-κBα.
T141 19999-20160 Sentence denotes The most abundant form of NF-κB is the p50–p65 heterodimer.26 The inactive form of NF-κB exists in the cytoplasm bound to an inhibitory complex containing I-κBα.
T8386 20161-20386 Sentence denotes Following cellular stimulation with LPS and chemotactic factors, NF-κB inducible kinase phosphorylates and activates the I-κB kinase (IKK) complex consisting of IKK-1 and IKK-2, which in turn phosphorylates the I-κBα subunit.
T142 20161-20386 Sentence denotes Following cellular stimulation with LPS and chemotactic factors, NF-κB inducible kinase phosphorylates and activates the I-κB kinase (IKK) complex consisting of IKK-1 and IKK-2, which in turn phosphorylates the I-κBα subunit.
T8387 20387-20495 Sentence denotes Phosphorylation, ubiquitination, and subsequent degradation of I-κBα results in NF-κB nuclear translocation.
T143 20387-20495 Sentence denotes Phosphorylation, ubiquitination, and subsequent degradation of I-κBα results in NF-κB nuclear translocation.
T8388 20496-20849 Sentence denotes This allows the transcription factor to bind to promoter regions of specific pro-inflammatory genes and influence transcription.27,28 Because a number of potential κB sites are present in the nucleotide sequences of the TNF-α gene, NF-κB is considered to be a critical transcriptional factor involved in TNF-α gene expression and protein synthesis.29,30
T144 20496-20849 Sentence denotes This allows the transcription factor to bind to promoter regions of specific pro-inflammatory genes and influence transcription.27,28 Because a number of potential κB sites are present in the nucleotide sequences of the TNF-α gene, NF-κB is considered to be a critical transcriptional factor involved in TNF-α gene expression and protein synthesis.29,30
T8389 20850-21146 Sentence denotes In this set of experiments, we demonstrated that PTX downregulates cytoplasmic I-κBα phosphorylation, nuclear NF-κB p65 phosphorylation/translocation, as well as NF-κB DNA-binding after LPS stimulation, suggesting that PTX exerts its function proximal to or at the level of I-κBα phosphorylation.
T145 20850-21146 Sentence denotes In this set of experiments, we demonstrated that PTX downregulates cytoplasmic I-κBα phosphorylation, nuclear NF-κB p65 phosphorylation/translocation, as well as NF-κB DNA-binding after LPS stimulation, suggesting that PTX exerts its function proximal to or at the level of I-κBα phosphorylation.
T8390 21147-21273 Sentence denotes Previous studies that have evaluated the effects of cAMP-elevating drugs on I-κBα and NF-κB have reported conflicting results.
T146 21147-21273 Sentence denotes Previous studies that have evaluated the effects of cAMP-elevating drugs on I-κBα and NF-κB have reported conflicting results.
T8391 21274-21745 Sentence denotes Haddad et al. reported results similar to ours with respect to the effects of PTX on I-κBα and NF-κB in pulmonary epithelial cells.31 Conversely, Neumann et al. proposed that increased intracellular cAMP stabilizes the interaction between I-κBα and NF-κB, therefore reducing NF-κB activation.32 In contrast, Takahashi et al. showed no difference in I-κBα degradation in Jurkat T-lymphocytes in the presence and absence of forskolin, an alternative cAMP inducing compound.
T147 21274-21745 Sentence denotes Haddad et al. reported results similar to ours with respect to the effects of PTX on I-κBα and NF-κB in pulmonary epithelial cells.31 Conversely, Neumann et al. proposed that increased intracellular cAMP stabilizes the interaction between I-κBα and NF-κB, therefore reducing NF-κB activation.32 In contrast, Takahashi et al. showed no difference in I-κBα degradation in Jurkat T-lymphocytes in the presence and absence of forskolin, an alternative cAMP inducing compound.
T8392 21746-22290 Sentence denotes This group also demonstrated that forskolin did not inhibit the DNA-binding activity of NF-κB and that CREB was not involved in forskolin-induced decrease in p65 transcriptional activity.33 Similar results were reported by Ollivier et al. using THP-1 cells and endothelial cells treated with forskolin or dibutyryl cAMP, two agents known to function through cAMP elevation and subsequent PKA activation.9 The differences between our results and those mentioned above may be explained by the use of different cell types and exogenous stimulants.
T148 21746-22290 Sentence denotes This group also demonstrated that forskolin did not inhibit the DNA-binding activity of NF-κB and that CREB was not involved in forskolin-induced decrease in p65 transcriptional activity.33 Similar results were reported by Ollivier et al. using THP-1 cells and endothelial cells treated with forskolin or dibutyryl cAMP, two agents known to function through cAMP elevation and subsequent PKA activation.9 The differences between our results and those mentioned above may be explained by the use of different cell types and exogenous stimulants.
T8393 22291-22547 Sentence denotes It is also possible that PTX exerts its anti-inflammatory effects on the NF-κB pathway independent of PKA, as suggested by the continued attenuation of LPS-induced TNF-α production by PTX in the presence or absence of PKA inhibition observed in this study.
T149 22291-22547 Sentence denotes It is also possible that PTX exerts its anti-inflammatory effects on the NF-κB pathway independent of PKA, as suggested by the continued attenuation of LPS-induced TNF-α production by PTX in the presence or absence of PKA inhibition observed in this study.
T8394 22548-22615 Sentence denotes This hypothesis is currently under investigation in our laboratory.
T150 22548-22615 Sentence denotes This hypothesis is currently under investigation in our laboratory.
T8395 22616-22775 Sentence denotes Furthermore, we utilized a broad PKA inhibitor in this study in addition to H89, but did not examine the effect of specific inhibition of various PKA isoforms.
T151 22616-22775 Sentence denotes Furthermore, we utilized a broad PKA inhibitor in this study in addition to H89, but did not examine the effect of specific inhibition of various PKA isoforms.
T8396 22776-22883 Sentence denotes This information may yield more insight into the mechanism by which PTX exerts its down-regulatory effects.
T152 22776-22883 Sentence denotes This information may yield more insight into the mechanism by which PTX exerts its down-regulatory effects.
T8397 22884-23056 Sentence denotes PDE inhibitors may also downregulate NF-κB transcriptional activity by altering the competitive binding of CREB and NF-κB to the promoter regions of pro-inflammatory genes.
T153 22884-23056 Sentence denotes PDE inhibitors may also downregulate NF-κB transcriptional activity by altering the competitive binding of CREB and NF-κB to the promoter regions of pro-inflammatory genes.
T8398 23057-23177 Sentence denotes Herein, we showed that the activation and DNA binding activity of CREB is upregulated in a dose–dependent manner by PTX.
T154 23057-23177 Sentence denotes Herein, we showed that the activation and DNA binding activity of CREB is upregulated in a dose–dependent manner by PTX.
T8399 23178-23418 Sentence denotes Of note, the increase in CREB activity was slightly alleviated by the addition of LPS, suggesting competition between PTX-induced CREB-associated gene transcription and those factors, such as NF-κB, which may be upregulated by LPS exposure.
T155 23178-23418 Sentence denotes Of note, the increase in CREB activity was slightly alleviated by the addition of LPS, suggesting competition between PTX-induced CREB-associated gene transcription and those factors, such as NF-κB, which may be upregulated by LPS exposure.
T8400 23419-23630 Sentence denotes This competition has been postulated to involve the recruitment of the co-activator CBP and its homologue p30034, suggesting an alternative mechanism that may be involved in the anti-inflammatory actions of PTX.
T156 23419-23630 Sentence denotes This competition has been postulated to involve the recruitment of the co-activator CBP and its homologue p30034, suggesting an alternative mechanism that may be involved in the anti-inflammatory actions of PTX.
T8401 23631-23712 Sentence denotes Future studies are planned to delineate the validity of this potential mechanism.
T157 23631-23712 Sentence denotes Future studies are planned to delineate the validity of this potential mechanism.
T8402 23713-23835 Sentence denotes The effects of PTX on other transcription factors, such as AP-1, c-fos, and c-jun, cannot be ruled out by our experiments.
T158 23713-23835 Sentence denotes The effects of PTX on other transcription factors, such as AP-1, c-fos, and c-jun, cannot be ruled out by our experiments.
T8403 23836-23981 Sentence denotes Activation of AP-1 and its components (c-fos and c-jun) by agents that enhance mononuclear cell activity has been implicated in TNF-α expression.
T159 23836-23981 Sentence denotes Activation of AP-1 and its components (c-fos and c-jun) by agents that enhance mononuclear cell activity has been implicated in TNF-α expression.
T8404 23982-24090 Sentence denotes Moreover, AP-1 and CREB recognize a similar DNA binding sequence in the promoter region of the TNF-α gene35.
T160 23982-24090 Sentence denotes Moreover, AP-1 and CREB recognize a similar DNA binding sequence in the promoter region of the TNF-α gene35.
T8405 24091-24290 Sentence denotes Given these relationships, it is conceivable that PTX may exert some of its anti-inflammatory actions by affecting AP-1 activation and favoring CREB binding, thus inhibiting TNF-α gene transcription.
T161 24091-24290 Sentence denotes Given these relationships, it is conceivable that PTX may exert some of its anti-inflammatory actions by affecting AP-1 activation and favoring CREB binding, thus inhibiting TNF-α gene transcription.
T8406 24291-24434 Sentence denotes Additional studies are necessary to elucidate the complex interactions between CREB, NF-κB, and AP-1 following LPS exposure and PDE inhibition.
T162 24291-24434 Sentence denotes Additional studies are necessary to elucidate the complex interactions between CREB, NF-κB, and AP-1 following LPS exposure and PDE inhibition.
T8407 24435-24571 Sentence denotes Furthermore, the effect of PTX on the cGMP pathway after LPS stimulation has not been explored, although we plan to do so in the future.
T163 24435-24571 Sentence denotes Furthermore, the effect of PTX on the cGMP pathway after LPS stimulation has not been explored, although we plan to do so in the future.
T8408 24572-24738 Sentence denotes In summary, we have demonstrated that PDE inhibition of human mononuclear cells downregulates TNF-α production, at least in part, through a PKA-independent mechanism.
T164 24572-24738 Sentence denotes In summary, we have demonstrated that PDE inhibition of human mononuclear cells downregulates TNF-α production, at least in part, through a PKA-independent mechanism.
T8409 24739-24916 Sentence denotes In addition, PTX attenuates the activity of NF-B while upregulating CREB activation after LPS stimulation, which may result in modulation of pro-inflammatory mediator synthesis.
T165 24739-24916 Sentence denotes In addition, PTX attenuates the activity of NF-B while upregulating CREB activation after LPS stimulation, which may result in modulation of pro-inflammatory mediator synthesis.
T8410 24917-25060 Sentence denotes Therefore, PTX may serve as a potential adjunct therapeutic for the treatment of conditions in which TNF-α production plays a significant role.
T166 24917-25060 Sentence denotes Therefore, PTX may serve as a potential adjunct therapeutic for the treatment of conditions in which TNF-α production plays a significant role.