Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis.

Inhibition by interferon-gamma of human mononuclear cell-mediated low density lipoprotein oxidation. Participation of tryptophan metabolism along the kynurenine pathway

In this study we examined the potential inhibition by interferon-gamma (IFN gamma) of the early stages of low density lipoprotein (LDL) oxidation mediated by human peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) in Ham's F-10 medium supplemented with physiological amounts of L-tryptophan (Trp). We assessed LDL oxidation by measuring the consumption of LDL's major antioxidant (i.e., alpha-tocopherol) and targets for oxidation (cholesteryllinoleate and cholesterylarachidonate), together with the accumulation of cholesterylester hydroperoxides and the increase in relative electrophoretic mobility of the lipoprotein particle. Exposure of PBMC or MDM to IFN gamma induced the degradation of extracellular Trp with concomitant accumulation of kynurenine, anthranilic and 3-hydroxyanthranilic acid (3HAA) in the culture medium. Formation of 3HAA, but neither Trp degradation nor formation of kynurenine and anthranilic acid, was inhibited by low amounts of diphenylene iodonium (DPI) in a concentration-dependent manner. In contrast to oxidative Trp metabolism, exposure of human PBMC or MDM to IFN gamma failed to induce degradation of arginine, and nitrite was not detected in the cell supernatant, indicating that nitric oxide synthase was not induced under these conditions. Incubation of LDL in Trp-supplemented F-10 medium resulted in a time-dependent oxidation of the lipoprotein that was accelerated in the presence of PBMC or MDM but inhibited strongly in the presence of both cells and IFN gamma, i.e., when Trp degradation and formation of 3HAA were induced. In contrast, when IFN gamma was added to PBMC or MDM in F-10 medium that was virtually devoid of Trp, inhibition of cell-accelerated LDL oxidation was not observed. Exogenous 3HAA added to PBMC or purified monocytes in the absence of IFN gamma also strongly and in a concentration-dependent manner inhibited LDL oxidation. Selective inhibition of IFN gamma-induced formation of 3HAA by DPI caused reversion of the inhibitory action of this cytokine on both PBMC- and MDM-mediated LDL oxidation. These results show that IFN gamma treatment of human PBMC or MDM in vitro attenuates the extent of LDL oxidation caused by these cells, and indicate that Trp degradation with formation of 3HAA is a major contributing factor to this inhibitory activity.

[Hyperinsulinemia as a consequence of systemic venous drainage in patients with pancreas transplantation]

To evaluate the metabolic consequences of pancreatic transplantation with systemic venous drainage on beta cell function, we examined insulin and C-peptide responses to arginine and secretin in type I diabetic recipients of pancreas transplantation (n = 16), and normal controls (n = 28). Basal insulin levels were 24 +/- 3 microU/l in pancreas recipients, and 7 +/- 1 microU/l in controls (p less than 0.001). Stimulated insulin levels following arginine (MANOVA, p less than 0.001), and secretin (MANOVA, p less than 0.001) were 1.5 to 3 fold elevated compared to controls. In contrast, integrated C-peptide responses following stimulation with arginine or secretin did not differ significantly between the two groups. We conclude that recipients of pancreas allografts with systemic venous drainage have elevated basal and stimulated insulin levels and that these alterations are primarily due to alterations of first pass hepatic insulin clearance although insulin resistance secondary to immunosuppressive therapy (including prednisone) may also play a contributing role. To avoid hyperinsulinemia and its possible long term adverse consequences, transplantation of pancreas allografts in sites with portal rather than systemic venous drainage may be preferable.

Systemic venous drainage of pancreas allografts as independent cause of hyperinsulinemia in type I diabetic recipients

To evaluate the metabolic consequences of pancreas transplantation with systemic venous drainage on beta-cell function, we examined insulin and C-peptide responses to glucose and arginine in type I (insulin-dependent) diabetic pancreas recipients (n = 30), nondiabetic kidney recipients (n = 8), and nondiabetic control subjects (n = 28). Basal insulin levels were 66 +/- 5 pM in control subjects, 204 +/- 18 pM in pancreas recipients (P less than 0.0001 vs. control), and 77 +/- 17 pM in kidney recipients. Acute insulin responses to glucose were 416 +/- 44 pM in control subjects, 763 +/- 91 pM in pancreas recipients (P less than 0.01 vs. control), and 589 +/- 113 pM in kidney recipients (NS vs. control). Basal and stimulated insulin levels in two pancreas recipients with portal venous drainage were normal. Integrated acute C-peptide responses were not statistically different (25.3 +/- 4.3 nM/min in pancreas recipients, 34.2 +/- 5.5 nM/min in kidney recipients, and 23.7 +/- 2.1 nM/min in control subjects). Similar insulin and C-peptide results were obtained with arginine stimulation, and both basal and glucose-stimulated insulin-C-peptide ratios in pancreas recipients were significantly greater than in control subjects. We conclude that recipients of pancreas allografts with systemic venous drainage have elevated basal and stimulated insulin levels and that these alterations are primarily due to alterations of first-pass hepatic insulin clearance, although insulin resistance secondary to immunosuppressive therapy (including prednisone) probably plays a contributing role. To avoid hyperinsulinemia and its possible long-term adverse consequences, transplantation of pancreas allografts into sites with portal rather than systemic venous drainage should be considered.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Roux-en-Y drainage of the pancreatic stump decreases pancreatic fistula after distal pancreatic resection

Clinically relevant fistula after distal pancreatic resection occurs in 5-30% of patients, prolonging recovery and considerably increasing in-hospital stay and costs. We tested whether routine drainage of the pancreatic stump into a Roux-en-Y limb after distal pancreatic resection decreased the incidence of fistula. From October 2001, data of all patients undergoing pancreatic distal resection were entered in a prospective database. From June 2003 after resection, the main pancreatic duct and the pancreatic stump were oversewn, and in addition, anastomosed into a jejunal Roux-en-Y limb by a single-layer suture (n = 23). A drain was placed near the anastomosis, and all patients received octreotide for 5-7 days postoperatively. The volume of the drained fluid was registered daily, and concentration of amylase was measured and recorded every other day. Patient demographics, hospital stay, pancreatic fistula incidence (> or =30 ml amylase-rich fluid/day on/after postoperative day 10), perioperative morbidity, and follow-up after discharge were compared with our initial series of patients (treated October 2001-May 2003) who underwent oversewing only (n = 20). Indications, patient demographics, blood loss, and tolerance of an oral diet were similar. There were four (20%) pancreatic fistulas in the "oversewn" group and none in the anastomosis group (p < 0.05). Nonsurgical morbidity, in-hospital stay, and follow-up were comparable in both groups.

Biosynthesis and expression of VE-cadherin is regulated by the PI3K/mTOR signaling pathway

Vascular endothelial (VE)-cadherin is an essential protein of adherens junctions of endothelial cells and plays a pivotal role in vascular homeostasis. Mammalian target of rapamycin complex 2 (mTORC2) deficient mice display defects in fetal vascular development. Blocking mTOR or the upstream kinase phosphoinositide 3-kinase (PI3K) led to a dose-dependently decrease of the VE-cadherin mRNA and protein expression. Immunofluorescent staining showed a strongly decreased expression of VE-cadherin at the interface of human umbilical endothelial cells (HUVECs) followed by intercellular gap formation. Herewith, we demonstrated that the expression of VE-cadherin is dependent on mTOR and PI3K signaling.

IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils

The contribution of basophils in allergic disease and other Th2-type immune responses depends on their persistence at sites of inflammation, but the ligands and molecular pathways supporting basophil survival are largely unknown. The comparison of rates of apoptosis and of the expression of antiapoptotic proteins in different human granulocyte types revealed that basophils have a considerably longer spontaneous life span than neutrophils and eosinophils consistent with high levels of constitutive Bcl-2 expression. Interleukin-3 (IL-3) is the only ligand that efficiently protects basophils from apoptosis as evidenced by screening a large number of stimuli. IL-3 up-regulates the expression of the antiapoptotic proteins cIAP2, Mcl-1, and Bcl-X(L) and induces a rapid and sustained de novo expression of the serine/threonine kinase Pim1 that closely correlates with cytokine-enhanced survival. Inhibitor studies and protein transduction of primary basophils using wild-type and kinase-dead Pim1-Tat fusion-proteins demonstrate the functional importance of Pim1 induction in the IL-3-enhanced survival. Our data further indicate that the antiapoptotic Pim1-mediated pathway operates independently of PI3-kinase but involves the activation of p38 MAPK. The induction of Pim1 leading to PI3-kinase-independent survival as described here for basophils may also be a relevant antiapoptotic mechanism in other terminally differentiated leukocyte types.

Regulation of Id1 expression by SRC: implications for targeting of the bone morphogenetic protein pathway in cancer

Deregulated activation of the Src tyrosine kinase and heightened Id1 expression are independent mediators of aggressive tumor biology. The present report implicates Src signaling as a critical regulator of Id1 gene expression. Microarray analyses showed that Id family genes were among the most highly down-regulated by incubation of A549 lung carcinoma cells with the small-molecule Src inhibitor AZD0530. Id1 transcript and protein levels were potently reduced in a dose-dependent manner concomitantly with the reduction of activated Src levels. These effects were conserved across a panel of lung, breast, prostate, and colon cancer cell lines and confirmed by the ability of PP2, Src siRNA, and Src-blocking peptides to suppress Id1 expression. PP2, AZD0530, and dominant-negative Src abrogated Id1 promoter activity, which was induced by constitutively active Src. The Src-responsive region of the Id1 promoter was mapped to a region 1,199 to 1,360 bps upstream of the translation start site and contained a Smad-binding element. Src was also required for bone morphogenetic protein-2 (BMP-2)-induced Id1 expression and promoter activity, was moderately activated by BMP-2, and complexed with Smad1/5. Conversely, Src inhibitors blocked Smad1/5 nuclear translocation and binding to the Src-responsive region of the Id1 promoter. Consistent with a role for Src and Id1 in cancer cell invasion, Src inhibitors and Id1 siRNA decreased cancer cell invasion, which was increased by Id1 overexpression. Taken together, these results reveal that Src positively interacts with the BMP-Smad-Id pathway and provide new ways for targeted inhibition of Id1.

Phosphatidylethanolamine in Trypanosoma brucei is organized in two separate pools and is synthesized exclusively by the Kennedy pathway

Phosphatidylethanolamine is a major phospholipid class of all eukaryotic cells. It can be synthesized via the CDP-ethanolamine branch of the Kennedy pathway, by decarboxylation of phosphatidylserine, or by base exchange with phosphatidylserine. The contributions of these pathways to total phosphatidylethanolamine synthesis have remained unclear. Although Trypanosoma brucei, the causative agent of human and animal trypanosomiasis, has served as a model organism to elucidate the entire reaction sequence for glycosylphosphatidylinositol biosynthesis, the pathways for the synthesis of the major phospholipid classes have received little attention. We now show that disruption of the CDP-ethanolamine branch of the Kennedy pathway using RNA interference results in dramatic changes in phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. By targeting individual enzymes of the pathway, we demonstrate that de novo phosphatidylethanolamine synthesis in T. brucei procyclic forms is strictly dependent on the CDP-ethanolamine route. Interestingly, the last step in the Kennedy pathway can be mediated by two separate activities leading to two distinct pools of phosphatidylethanolamine, consisting of predominantly alk-1-enyl-acyl- or diacyl-type molecular species. In addition, we show that phosphatidylserine in T. brucei procyclic forms is synthesized exclusively by base exchange with phosphatidylethanolamine.

Activation of the epidermal growth factor receptor (EGFR) by a novel metalloprotease pathway

Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFalpha and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFkappaB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production.

Statins induce regulatory T cell recruitment via a CCL1 dependent pathway

The statins, a group of inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A reductase, are reported to influence a variety of immune system activities through 3-hydroxy-3-methylglutaryl coenzyme A reductase-dependent and -independent mechanisms. How statin treatment regulates immune system function in vivo nonetheless remains to be fully defined. We analyzed the immunomodulatory effects of lovastatin in a Candida albicans-induced delayed-type hypersensitivity reaction in mice. In this model, lovastatin administration reduced the acute inflammatory response elicited by C. albicans challenge. This anti-inflammatory activity of lovastatin was associated with a shift from a Th1 to a Th2 immune response, as well as an increase in the percentage of regulatory T cells at the inflammation site and in the regional draining lymph node. The lovastatin-induced increase in regulatory T cells in the inflamed skin was dependent on expression of CCL1, a chemokine that is locally up-regulated by statin administration. The anti-inflammatory effect of lovastatin was abrogated in CCL1-deficient mice. These results suggest that local regulation of chemokine expression may be an important process in statin-induced modulation of the immune system.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.