Biomedical subjects

James A Cook

Publications and source records attributed to James A Cook.

At least 19 recordsLinked to original sources

PubMed2006

Gi proteins regulate lipopolysaccharide and Staphylococcus aureus induced cytokine production but not (1--> 3)-beta-D-glucan induced cytokine suppression.

Previous studies have demonstrated that bacterial lipopolysaccharide (LPS) and heat killed Staphylococcus aureus (SA) activation of inflammatory cells depended in part upon activation of heterotrimeric Gi proteins. It has also been shown that (1 --> 3) beta-D-glucan can suppress inflammatory cell activation by microbial products although the cellular mechanism of the glucan effect remains to be clearly defined. We hypothesized that Gi proteins function as a common convergent signaling pathway for both LPS and SA leading to monocyte mediator production. Additionally, we hypothesized that soluble glucan suppresses LPS and SA induced cytokine production via Gi protein coupled signaling. Human THP-1 promonocytic cells were pretreated with pertussis toxin (PTx, 100 ng/ml or 1 microgram/ml) 6 hours prior to stimulation with LPS (10 microgram/ml) and SA (10 microgram/ml) and/or soluble glucan (10 microgram/ml). Both LPS and SA significantly (p < 0.05) induced cytokine production IL-6 > TNF alpha > IL-1 beta > GM-CSF > IL-10 > IFN gamma. The induction of these cytokines was significantly (p < 0.05) suppressed by PTx. Glucan treatment alone had no effect on cytokine production but suppressed (P < 0.05) LPS and SA induced cytokines. PTx further augmented (p > 0.05) the inhibitory effect of glucan on the LPS and SA induced cytokine expression. The data support the hypothesis that Gi proteins function as a common signaling protein for both LPS and SA induction of pro-and anti-inflammatory cytokines and that soluble glucan effectively suppresses cytokine production to the microbial stimuli. In contrast, the effect of soluble glucan on inhibiting cellular activation by LPS and SA is Gi protein independent.

Cell Culture Techniques↗

PubMed2006

Differential regulation of lipopolysaccharide and Gram-positive bacteria induced cytokine and chemokine production in splenocytes by Galphai proteins.

Heterotrimeric Gi proteins play a role in lipopolysaccharide (LPS) and Staphylococcus aureus (SA) activated signaling leading to inflammatory mediator production. We hypothesized that genetic deletion of Gi proteins would alter cytokine and chemokine production induced by LPS and SA. LPS- and heat killed SA-induced cytokine and chemokine production in splenocytes from wild type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS- or SA-induced production of TNFalpha, IL-6, IFNgamma, IL-12, IL-17, GM-CSF, MIP-1alpha, MCP-1, MIG and IP-10 were significantly increased (1.2 to 33 fold, p<0.05) in splenocytes harvested from Galpha(i2)(-/-) mice compared with WT mice. The effect of Galpha(i) protein depletion was remarkably isoform specific. In splenocytes from Galpha(i1/3) (-/-) mice relative to WT mice, SA-induced IL-6, IFNgamma, GM-CSF, and IP-10 levels were decreased (59% to 86%, p<0.05), whereas other LPS- or SA-stimulated cytokines and chemokines were not different relative to WT mice. LPS- and SA-induced production of KC were unchanged in both groups of the genetic deficient mice. Splenocytes from both Galpha(i2) (-/-) and Galpha(i1/3) (-/-) mice did not exhibit changes in TLR2 and TLR4 expression. Also analysis of splenic cellular composition by flow cytometry demonstrated an increase in splenic macrophages and reduced CD4 T cells in both Galpha(i2) (-/-) and Galpha(i1/3) (-/-) mice relative to WT mice. The disparate response of splenocytes from the Galpha(i2) (-/-) relative to Galpha(i1/3) (-/-) mice therefore cannot be attributed to major differences in spleen cellular composition. These data demonstrate that G(i2) and G(i1/3) proteins are both involved and differentially regulate splenocyte inflammatory cytokine and chemokine production in a highly Gi isoform specific manner in response to LPS and Gram-positive microbial stimuli.

Animals↗

PubMed2006

Synthesis of fluorescent derivatives of wortmannin and demethoxyviridin as probes for phosphatidylinositol 3-kinase.

Fluorescent analogs were synthesized of the potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin. The esterification of 11-deacetylwortmannin, 17-hydroxywortmannin, and demethoxyviridin with the fluorescent carboxylic acids NBD-sarcosine and 7-dimethylaminocoumarin-4-acetic acid generated six novel fluorescent esters. Potent inhibition of PI 3-kinase-alpha was observed for the derivatives of 11-desacetylwortmannin and demethoxyviridin.

Androstadienes↗

PubMed2006

Synergistic effect of peroxisome proliferator activated receptor-gamma and liver X receptor-alpha in the regulation of inflammation in macrophages.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) and liver X receptor-alpha (LXRalpha) are nuclear ligand-activated transcription factors, which regulate lipid metabolism and inflammation. Murine J774.2 macrophages were stimulated with Escherichia coli lipopolysaccharide (concentration, 10 microg/mL) with or without the PPARgamma ligand, 15-deoxy-Delta prostaglandin J2 (15d-PGJ2), or the LXRalpha ligands, 22(R)-hydroxycholesterol and T0901317 (concentration range, 0.01-10 micromol/L), alone or in combination. Nitric oxide (NO) metabolites and tumor necrosis factor alpha production, inducible NO synthase expression, and mitochondrial respiration were measured. When added to the cells as single agents, 15d-PGJ2, 22(R)-hydroxycholesterol, or T0901317 reduced the lipopolysaccharide-induced NO and tumor necrosis factor alpha production and the inducible NO synthase expression, and partially maintained mitochondrial respiration in a concentration-dependent manner. When added to the cells in combination at suboptimal concentrations, 15d-PGJ2 with 22(R)-hydroxycholesterol, or 15d-PGJ2 with T0901317, exerted anti-inflammatory effects similar to much higher concentrations (10,000-fold to 100,000-fold) of each ligand alone. The anti-inflammatory effects of these ligands, alone or in combination, were associated with reduction of nuclear factor-kappaB activation and with enhancement of PPARgamma DNA binding. LXRalpha expression was upregulated in response to 15d-PGJ2 and to the LXRalpha ligands when added alone or in combination. Immunoprecipitation experiments revealed that PPARgamma interacted with LXRalpha. Our data demonstrate that the PPARgamma ligand, 15d-PGJ2, and the LXRalpha ligands, 22(R)-hydroxycholesterol and T0901317, although binding to different nuclear receptors (i.e., PPARgamma and LXRalpha, respectively), affect mediator production through common cell signaling events and exert a synergistic potentiation in a combined treatment at suboptimal concentrations. Thus, our data suggest that PPARgamma and LXRalpha may interact in controlling the inflammatory response in macrophages.

Animals↗

PubMed2006

The phosphatidylinositol 3 kinase pathway regulates tolerance to lipopolysaccharide and priming responses to Staphylococcus aureus and lipopolysaccharide.

Our previous studies have demonstrated that although LPS and Staphylococcus aureus induce homologous tolerance, they induce priming to each other instead of cross-tolerance. The phosphatidylinositol 3 (PI3) kinase pathway has been implicated in microbial signaling and inflammatory gene expression regulation. We hypothesized that LPS or S. aureus induced tolerance and priming responses to each other are PI3 kinase pathway-dependent. CD1 mice received intraperitoneal injections of 1% Biogel and were treated intraperitoneally with vehicle, LPS, or S. aureus (5 mg/kg) 3 days later. Peritoneal macrophages (MØ) were harvested 24 h later and exposed to vehicle or the PI3 kinase inhibitors wortmannin (10 nmol/L) or LY294002 (10 nmol/L) 1 h before in vitro stimulation with LPS or S. aureus (10 microg/mL). Both LPS and S. aureus significantly induced tumor necrosis factor alpha and thromboxane B2 synthesis (P < 0.05, n = 3) in naive cells. LPS and S. aureus induced homologous tolerance were associated with suppressed tumor necrosis factor alpha and thromboxane B2 levels but augmented interleukin 10 production. However, LPS and S. aureus induced priming to each other, as shown by augmented mediator production. Wortmannin and LY294002 reversed LPS tolerance yet had no effect on S. aureus tolerance. PI3 kinase blockade attenuated the priming responses to both LPS and S. aureus. Mice pretreated with LPS and challenged with LPS were protected. In contrast, mice pretreated with LPS and wortmannin demonstrated LPS tolerance reversal. These data suggest that PI3 kinase is essential for LPS induced homologous tolerance and reciprocal LPS and S. aureus induced priming responses.

Androstadienes↗

PubMed2005

Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins.

Heterotrimeric G(i) proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G(i) proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Galpha(i2) or Galpha(i1/3) protein in Galpha(i2)-knockout (Galpha(i2)-/-) or Galpha(i1/3)-knockout (Galpha(i1/3)-/-) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (MPhi) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF-alpha and IFN-gamma in splenocytes from Galpha(i2)-/- mice compared with wild-type (WT) mice. Also, LPS-induced TNF-alpha was increased in splenocytes from Galpha(i1/3)-/- mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-alpha, IL-10, and thromboxane B(2) (TxB(2)) production was decreased in MPhi harvested from Galpha(i2)-/- mice. Also, LPS-induced production of IL-10 and TxB(2) was decreased in MPhi from Galpha(i1/3)-/- mice. In subsequent in vivo studies, TNF-alpha levels after LPS challenge were significantly greater in Galpha(i2)-/- mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Galpha(i2)-/- mice compared with WT mice. These data suggest that G(i) proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli.

Animals↗

PubMed2005

The development of potent and selective bisarylmaleimide GSK3 inhibitors.

Many 3-aryl-4-(1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl)maleimides exhibit potent GSK3 inhibitory activity (<100 nM IC(50)), although few show significant selectivity (>100x) versus CDK2, CDK4, or PKCbetaII. However, combining 3-(imidazo[1,2-a]pyridin-3-yl), 3-(pyrazolo[1,5-a]pyridin-3-yl) or aza-analogs with a 4-(2-acyl-(1,2,3,4-tetrahydro[1,4]diazepino[6,7,1-hi]indol-7-yl)) group on the maleimide resulted in very potent inhibitors of GSK3 (</=5 nM) with >160 to >10,000-fold selectivity versus CDK2/4 and PKCbetaII. These compounds also inhibited tau phosphorylation in cells and were effective in lowering plasma glucose in a rat model of type 2 diabetes (ZDF rat).

Animals↗

PubMed2005

Eicosanoids.

Explore the source record for details and available documents.

Animals↗

PubMed2005

Peroxisome proliferator-activated receptor-gamma is a new therapeutic target in sepsis and inflammation.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear receptor superfamily and a ligand-activated transcription factor with pleiotropic effects on lipid metabolism, inflammation, and cell proliferation. PPARgamma forms a heterodimer with the retinoid X receptor and upon ligand-activation binds to the PPAR response element in the promoter of genes to allow transcription. The class of insulin-sensitizing drugs known as thiazolidinediones have been identified as specific PPARgamma agonists that have allowed the characterization of many genes regulated by PPARgamma. Thiazolidinediones include rosiglitazone, pioglitazone, troglitazone, and ciglitazone. In addition to these synthetic agonists, cyclopentenone prostaglandins of the J2 series have been identified as natural ligands for PPARgamma. Several in vitro and in vivo studies have demonstrated that pharmacological activation of PPARgamma by 15-deoxy-Delta(12,14)-PGJ2 (15d-PGJ2) or thiazolidinediones has anti-inflammatory effects. This article provides an overview of the role of PPARgamma in regulating the inflammatory response and emphasizes the potential efficacy of PPARgamma ligands as novel therapeutic approaches beyond diabetes in sepsis, inflammation, and reperfusion injury.

Animals↗

PubMed2005

15-Deoxy-delta(12,14)-prostaglandin J(2) (15D-PGJ(2)), a peroxisome proliferator activated receptor gamma ligand, reduces tissue leukosequestration and mortality in endotoxic shock.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that requires ligand activation for transcription. Experimental studies have shown that 15-deoxy-Delta-PGJ2 (15d-PGJ2) is a natural PPARgamma ligand which has potent anti-inflammatory properties. This study was designed to examine the effect and the molecular mechanisms of 15d-PGJ2 on tissue neutrophil infiltration and survival in endotoxic shock. Male Swiss albino mice were subjected to intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS, 25 mg/kg). Three hours after LPS mice received vehicle or 15d-PGJ2 (1 mg/kg) and continued treatment every 12 hours. Survival was monitored for 72 hours. In a separate experiment, mice were sacrificed 6 hours after LPS and tissue examined. In vehicle-treated mice, LPS injection resulted in a survival rate of 9%. Marked lung injury was characterized by hemorrhage, infiltration of inflammatory cells and reduction of alveolar space. Elevated levels of myeloperoxidase activity in lung and small intestine were indicative of infiltration of neutrophils. Increased expression of intercellular adhesion molecule-1, vascular cellular adhesion molecule-1 and E-selectin were observed in the lung and small intestine. These inflammatory events were associated with reduced expression of PPARgamma and with activation of nuclear factor-kappaB (NF-kappaB) in the lung. Treatment with 15d-PGJ2 improved survival rate to 55%, downregulated expression of adhesion molecules and reduced neutrophil infiltration in tissues. These beneficial effects were associated with reduced activation of NF-kappaB DNA binding, whereas expression and DNA binding of PPARgamma and expression of the cytoprotective heat shock protein (HSP) 70 were increased in the lung. Our data demonstrate that 15d-PGJ2 ameliorates endotoxic shock most likely through repressing the proinflammatory pathway of NF-kappaB and enhancement of the cytoprotective heat shock response.

Animals↗

PubMed2004

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming.

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.

Chemokines↗

PubMed2004

Cloning, expression, purification, and characterization of the human Class Ia phosphoinositide 3-kinase isoforms.

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.

Animals↗

PubMed2004

Staphylococcus aureus and lipopolysaccharide induce homologous tolerance but heterologous priming: role of interferon-gamma.

Lipopolysaccharide (LPS), the gram-negative bacterial cell wall component, induces tolerance to a secondary challenge of LPS in macrophages (Mphi) as evidenced by reduced inflammatory mediator production. However, it is uncertain if heat-killed (HK) gram-positive bacteria Staphylococcus aureus (Sa) can induce a similar tolerance and alter responses to LPS. We hypothesized that HKSa induces homologous tolerance and cross tolerance to LPS stimulation in human promonocytic THP-1 cells. We measured TNF-alpha, TxB2, and IFN-gamma production and the phosphorylation of p38, JNK, and ERK-1/2 in human promonocytic THP-1 cells. HKSa (10 microg/mL) significantly stimulated naive (nonpretreated) cell TNF-alpha (P<0.05) and TxB2 production (P<0.05). However, HKSa-pretreated cells challenged secondarily with HKSa (10 microg/mL) exhibited a decrease in the production of TNF-alpha (89 +/- 5%, P<0.05) and TxB2 (85 +/- 3%, P<0.05) compared with HKSa-stimulated naive cells. By contrast, secondary LPS challenge of HKSa-pretreated cells augmented TNF-alpha (41 +/- 3%, P<0.05) and TxB2 (42 +/- 6%, P<0.05) compared with LPS-stimulated naive cells. In naive cells, HKSa and LPS stimulation also significantly phosphorylated the mitogen-activated kinases (MAPKs) p38, JNK, and ERK-1/2 (P<0.005) compared with basal levels. HKSa and LPS induced homologous tolerance as evidenced by the down-regulation of the three MAPK (P<0.05), thus paralleling data on mediator production. HKSa-pretreated cells' priming responses to LPS correlated with augmented phosphorylation of JNK and p38 (P<0.05), whereas ERK-1/2 phosphorylation remained down-regulated. In contrast to TNF-alpha and TxB2 production, HKSa-induced IFN-gamma was up-regulated (26 +/- 5%) in HKSa-pretreated cells compared with HKSa-stimulated naive cells. IFN-gamma antibody exhibited reversed priming in HKSa-pretreated cells as evidenced by a reduction in TNF-alpha. Exogenous human IFN-gamma- (1 microg/mL) and HKSa-pretreated cells secondarily stimulated with HKSa did not prevent the induction of tolerance. In contrast, exogenous IFN-gamma pretreatment prevented the induction of LPS homologous tolerance resulting in an increase in TNF-alpha production. The data demonstrate that HKSa induces homologous tolerance but causes priming to LPS.

Blotting, Western↗

PubMed2004

Toll-like receptor 4 coupled GI protein signaling pathways regulate extracellular signal-regulated kinase phosphorylation and AP-1 activation independent of NFkappaB activation.

Previous studies have implicated heterotrimeric Gi proteins in signaling leading to inflammatory mediator production induced by lipopolysaccharide (LPS). TLR4 has recently been shown to play a central role in response to LPS activation. We hypothesized that Gi proteins are coupled to TLR4 activation of signaling pathways. To inhibit Gi protein function, human embryonic kidney (HEK) 293 cells or RAW 264.7 cells were pretreated with pertussis toxin (PTx), an inhibitor of receptor-Galphai interaction, or transfected with dominant negative Galphai3 (Galphai3dn) or Galphai2 minigene (an inhibitory carboxyl terminus of Galphai2) plasmid. The cells were subsequently transfected with constitutively active TLR4 (TLR4ca) plasmid or TLR4ca together with an NFkappaB or AP-1 reporter construct. TLR4ca transfection induced ERK 1/2 activation (157 +/- 14%, P < 0.01), AP-1 activation (4.0 +/- 0.2-fold, P < 0.01), and NFkappaB activation (8.1 +/- 0.4-fold, P < 0.01) compared with empty vector controls. Pretreatment with PTx inhibited TLR4ca-induced ERK 1/2 phosphorylation (30 +/- 7%, P < 0.05) and AP-1 activation (36 +/- 3%, P < 0.05) but did not inhibit NFkappaB activation. Cotransfection of TLR4ca with Galphai3dn or Galphai2 minigene also reduced TLR4ca-induced ERK 1/2 phosphorylation (34 +/- 10% and 33 +/- 5%, respectively, P < 0.05). Constitutively active Galphai2 and Galphai3 plasmids potentiated TLR4ca-induced ERK 1/2 phosphorylation (27 +/- 3% and 41 +/- 6%, respectively, P < 0.05). betaARK-ct plasmid, which inhibits the function of betagamma subunit of G protein, has no effect on TLR4ca-induced ERK 1/2 phosphorylation. These data support our hypothesis and provide the first evidence that Galphai-coupled signaling pathways are activated by TLR4. The TLR4-activated Galphai signaling pathway activates ERK 1/2 phosphorylation and AP-1 activation independently of TLR4-mediated signaling to NFkappaB activation.

Cell Line↗

PubMed2004

Molecular mechanisms of endotoxin tolerance.

The phenomenon of endotoxin tolerance has been widely investigated, but to date, the molecular mechanisms of endotoxin tolerance remain to be resolved clearly. The discovery of the Toll-like receptor (TLR) family as the major receptors for lipopolysaccharide (LPS) and other bacterial products has prompted a resurgence of interest in endotoxin tolerance mechanisms. Changes of cell surface molecules, signaling proteins, pro-inflammatory and anti-inflammatory cytokines and other mediators have been examined. During tolerance expression of LPS-binding protein (LBP), CD14, myeloid differentiation protein-2 (MD-2) and TLR2 are unchanged or up-regulated, whereas TLR4 is transiently suppressed or unchanged. Proximal post-receptor signaling proteins that are altered in tolerance include augmented degradation of interleukin-1 receptor-associated kinase (IRAK), and decreased TLR4-myeloid differentiation factor 88 (MyD88) and IRAK-MyD88 association. Tolerance has also been shown to be associated with decreased Gi protein content and activity, decreased protein kinase C (PKC) activity, reduction in mitogen-activated protein kinase (MAP kinase) activity, and reduced activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB) induced gene transactivation. However, not all signaling proteins and pathways are suppressed in tolerance and induction of specific anti-inflammatory proteins and signaling pathways may serve important counter inflammatory functions. The latter include induction of IRAK-M and suppressor of cytokine-signaling-1 (SOCS-1), phosphoinositide-3-kinase (PI3K) signaling, and increased or maintained expression of inhibitor-kappaB (IkappaB) isoforms. Also at the nuclear level, increase in the NF-kappaB subunit p50 homodimer expression and increased activation of peroxisome-proliferator-activated receptors-gamma (PPARgamma) have been linked to tolerance phenotype. Although there are species and cellular variations in manifestation of the LPS tolerant phenotype, it is clear that the tolerance phenomena have evolved as a complex orchestrated counter regulatory response to inflammation.

Animals↗

PubMed2003

Peroxisome proliferator activator receptor-gamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J2 and ciglitazone, reduce systemic inflammation in polymicrobial sepsis by modulation of signal transduction pathways.

Peroxisome proliferator activator receptor-gamma (PPARgamma) is a nuclear receptor that controls the expression of several genes involved in metabolic homeostasis. We investigated the role of PPARgamma during the inflammatory response in sepsis by the use of the PPARgamma ligands, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and ciglitazone. Polymicrobial sepsis was induced by cecal ligation and puncture in rats and was associated with hypotension, multiple organ failure, and 50% mortality. PPARgamma expression was markedly reduced in lung and thoracic aorta after sepsis. Immunohistochemistry showed positive staining for nitrotyrosine and poly(ADP-ribose) synthetase in thoracic aortas. Plasma levels of TNF-alpha, IL-6, and IL-10 were increased. Elevated activity of myeloperoxidase was found in lung, colon, and liver, indicating a massive infiltration of neutrophils. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex, and c-Jun NH(2)-terminal kinase and, subsequently, activation of NF-kappaB and AP-1 in the lung. In vivo treatment with ciglitazone or 15d-PGJ(2) ameliorated hypotension and survival, blunted cytokine production, and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of the PPARgamma ligands were associated with the reduction of IkappaB kinase complex and c-Jun NH(2)-terminal kinase activation and the reduction of NF-kappaB and AP-1 DNA binding in the lung. Furthermore, treatment with ciglitazone or 15d-PGJ(2) up-regulated the expression of PPARgamma in lung and thoracic aorta and abolished nitrotyrosine formation and poly(ADP-ribose) expression in aorta. Our data suggest that PPARgamma ligands attenuate the inflammatory response in sepsis through regulation of the NF-kappaB and AP-1 pathways.

Animals↗

PubMed2003

Demonstrating the intrinsic ion channel activity of virally encoded proteins.

This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.

Animals↗

PubMed2003

Involvement of G(i) proteins and Src tyrosine kinase in TNFalpha production induced by lipopolysaccharide, group B Streptococci and Staphylococcus aureus.

Previous studies have suggested that heterotrimeric G(i) proteins, Src tyrosine kinase and phosphatidylinositol-3 kinase (PI3 Kinase) are involved in signaling events induced by lipopolysaccharide (LPS) leading to pro-inflammatory cytokines gene expression. To investigate the involvement of these mediators in Gram-positive bacteria induced pro-inflammatory cytokine expression, LPS (10 ng/ml), heat killed group B Streptococci (GBS 1 microg/ml) and Staphylococcus aureus (SA 10 microg/ml) were used to induce TNFalpha production in the murine J774A.1 macrophage (MØ) cell line and human promonocytic THP-1 cell line. Pertussis toxin (PTx, 1 microg/ml), an inhibitor of G(i) protein; pyrazolopyrimidine-2 (PP2, 1 or 25 microM), a Src tyrosine kinase inhibitor; and LY294002 (100 nM), an inhibitor of PI3 Kinase were used to examine the involvement of G(i), Src tyrosine kinase and PI3 Kinase, respectively, in TNFalpha production. In J774A.1 cells, pretreatment with PTx and PP2 attenuated TNFalpha production induced by LPS (60+/-9% and 81+/-11% inhibition, n=3, p<0.05, respectively), GBS (95+/-1% and 80+/-6% inhibition, n=3, p<0.05, respectively) and SA (51+/-18% and 68+/-16% inhibition, n=4, p<0.05, respectively). However, pretreatment with LY 294002 inhibited LPS induced TNFalpha production (82+/-13% inhibition, n=3, p<0.05), but did not inhibit GBS or SA induced TNFalpha production. In THP-1 cells, pretreatment with PTx, PP2 and LY 294002 inhibited TNFalpha production induced by LPS (84+/-3%, 59+/-12% and 84+/-4% inhibition, n=3, p<0.05, respectively) and SA (56+/-7%, 87+/-1% and 35+/-6% inhibition, n=3, p<0.05, respectively). These data support our hypothesis that G(i)-coupled and Src tyrosine kinase-coupled signaling pathways are involved in both Gram-negative and Gram-positive bacteria induced pro-inflammatory cytokine expression. However, unlike LPS, involvement of PI3 Kinase in Gram-positive bacteria induced signaling pathways are species dependent.

Animals↗