Transduction patterns in the CNS following various routes of AAV-5-mediated gene delivery.

Various administration routes of adeno-associated virus (AAV)-based gene therapy have been examined to target the central nervous system to answer the question what the most optimal delivery route is for treatment of the brain with certain indications. In this study, we evaluated AAV5 vector system for its capability to target the central nervous system via intrastriatal, intrathalamic or intracerebroventricular delivery routes in rats. AAV5 is an ideal candidate for gene therapy because of its relatively low level of existing neutralizing antibodies compared to other serotypes, and its broad tissue and cell tropism. Intrastriatal administration of AAV5-GFP resulted in centralized localized vector distribution and expression in the frontal part of the brain. Intrathalamic injection showed transduction and gradient expression from the rostral brain into lumbar spinal cord, while intracerebroventricular administration led to a more evenly, albeit relatively superficially distributed, transduction and expression throughout the central nervous system. To visualize the differences between localized and intra-cerebral spinal fluid administration routes, we compared intrastriatal to intracerebroventricular and intrathecal administration of AAV5-GFP. Together, our results demonstrate that for efficient transgene expression, various administration routes can be applied.

AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington's disease.

Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. To date, there is no treatment to halt or reverse the course of HD. Lowering of either total or only the mutant HTT expression is expected to have therapeutic benefit. This can be achieved by engineered micro (mi)RNAs targeting HTT transcripts and delivered by an adeno-associated viral (AAV) vector. We have previously showed a miHTT construct to induce total HTT knock-down in Hu128/21 HD mice, while miSNP50T and miSNP67T constructs induced allele-selective HTT knock-down in vitro. In the current preclinical study, the mechanistic efficacy and gene specificity of these selected constructs delivered by an AAV serotype 5 (AAV5) vector was addressed using an acute HD rat model. Our data demonstrated suppression of mutant HTT messenger RNA, which almost completely prevented mutant HTT aggregate formation, and ultimately resulted in suppression of DARPP-32-associated neuronal dysfunction. The AAV5-miHTT construct was found to be the most efficient, although AAV5-miSNP50T demonstrated the anticipated mutant HTT allele selectivity and no passenger strand expression. Ultimately, AAV5-delivered-miRNA-mediated HTT lowering did not cause activation of microglia or astrocytes suggesting no immune response to the AAV5 vector or therapeutic precursor sequences. These preclinical results suggest that using gene therapy to knock-down HTT may provide important therapeutic benefit for HD patients and raised no safety concerns, which supports our ongoing efforts for the development of an RNA interference-based gene therapy product for HD.

Therapeutic expression of hairpins targeting apolipoprotein B100 induces phenotypic and transcriptome changes in murine liver.

Constitutive expression of short hairpin RNAs (shRNAs) may cause cellular toxicity in vivo and using microRNA (miRNA) scaffolds can circumvent this problem. Previously, we have shown that embedding small interfering RNA sequences targeting apolipoprotein B100 (ApoB) in shRNA (shApoB) or miRNA (miApoB) scaffolds resulted in differential processing and long-term efficacy in vivo. Here we show that adeno-associated virus (AAV)-shApoB- or AAV-miApoB-mediated ApoB knockdown induced differential liver morphology and transcriptome expression changes. Our analyses indicate that ApoB knockdown with both shApoB and miApoB resulted in alterations of genes involved in lipid metabolism. In addition, in AAV-shApoB-injected animals, genes involved in immune system activation or cell growth and death were affected, which was associated with increased hepatocyte proliferation. Subsequently, in AAV-miApoB-injected animals, changes of genes involved in oxidoreductase activity, oxidative phosphorylation and nucleic bases biosynthetic processes were observed. Our results demonstrate that long-term knockdown of ApoB in vivo by shApoB or miApoB induces several transcriptome changes in murine liver. The increased hepatocyte profileration by AAV-shRNA may have severe long-term effects indicating that AAV-mediated RNA interference therapy using artificial miRNA may be a safer approach for familial hypercholesterolemia therapy.

Efficacy and long-term safety of alipogene tiparvovec (AAV1-LPLS447X) gene therapy for lipoprotein lipase deficiency: an open-label trial.

We describe the 2-year follow-up of an open-label trial (CT-AMT-011-01) of AAV1-LPL(S447X) gene therapy for lipoprotein lipase (LPL) deficiency (LPLD), an orphan disease associated with chylomicronemia, severe hypertriglyceridemia, metabolic complications and potentially life-threatening pancreatitis. The LPL(S447X) gene variant, in an adeno-associated viral vector of serotype 1 (alipogene tiparvovec), was administered to 14 adult LPLD patients with a prior history of pancreatitis. Primary objectives were to assess the long-term safety of alipogene tiparvovec and achieve a ≥40% reduction in fasting median plasma triglyceride (TG) at 3-12 weeks compared with baseline. Cohorts 1 (n=2) and 2 (n=4) received 3 × 10(11) gc kg(-1), and cohort 3 (n=8) received 1 × 10(12) gc kg(-1). Cohorts 2 and 3 also received immunosuppressants from the time of alipogene tiparvovec administration and continued for 12 weeks. Alipogene tiparvovec was well tolerated, without emerging safety concerns for 2 years. Half of the patients demonstrated a ≥40% reduction in fasting TG between 3 and 12 weeks. TG subsequently returned to baseline, although sustained LPL(S447X) expression and long-term changes in TG-rich lipoprotein characteristics were noted independently of the effect on fasting plasma TG.

Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues.

A cDNA clone of murine macrophage inflammatory protein 2 (MIP-2) has been isolated from a library prepared from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the nucleotide sequence determined. This cDNA was used to clone cDNAs for human homologues of MIP-2 from a library prepared from phorbol myristate acetate-treated and LPS-stimulated U937 cells. Two homologues were isolated and sequenced. Human MIP-2 alpha and MIP-2 beta are highly homologous to each other and to a previously isolated gene, human gro/melanoma growth-stimulating activity (MGSA). These three human genes, MIP-2 alpha, MIP-2 beta, and gro/MGSA, constitute a sub-family within the cytokine family represented by platelet factor 4 and interleukin 8.

Regulation of interleukin 10 release by tumor necrosis factor in humans and chimpanzees.

Interleukin 10 (IL-10) has been shown to inhibit endotoxin-induced tumor necrosis factor (TNF) production. To assess the role of TNF in the induction of IL-10 in endotoxemia, four healthy men were studied after a bolus intravenous injection of recombinant human TNF (50 micrograms/m2). In addition, 13 healthy chimpanzees were investigated after a bolus intravenous injection of Escherichia coli endotoxin (4 ng/kg), 6 animals received endotoxin only, 4 animals received a simultaneous intravenous injection of a monoclonal anti-TNF antibody, whereas 3 chimpanzees were treated with an anti-TNF F(ab')2 fragment 30 min after the administration of endotoxin. TNF induced a modest rise in IL-10 concentrations peaking after 45 min (47 +/- 32 pg/ml; p < 0.05). IL-10 peaked 2 h after injection of endotoxin (202 +/- 61 pg/ml; p < 0.005). In both anti-TNF-treated groups, the early endotoxin-induced TNF activity was completely neutralized. Simultaneous anti-TNF treatment attenuated endotoxin-induced IL-10 release (73 +/- 13 pg/ml; p < 0.01 versus endotoxin alone), whereas postponed anti-TNF treatment did not significantly affect this response (p = 0.21). These results indicate that TNF, in part, mediates the induction of IL-10 in endotoxemia, resulting in an autoregulatory feedback loop.

Antiinflammatory effects of reconstituted high-density lipoprotein during human endotoxemia.

High-density lipoprotein (HDL) has been found to neutralize LPS activity in vitro and in animals in vivo. We sought to determine the effects of reconstituted HDL (rHDL) on LPS responsiveness in humans in a double-blind, randomized, placebo-controlled, cross-over study. rHDL, given as a 4-h infusion at 40 mg/kg starting 3.5 h before endotoxin challenge (4 ng/kg), reduced flu-like symptoms during endotoxemia, but did not influence the febrile response. rHDL potently reduced the endotoxin-induced release of TNF, IL-6, and IL-8, while only modestly attenuating the secretion of proinflammatory cytokine inhibitors IL-1ra, soluble TNF receptors and IL-10. In addition, rHDL attenuated LPS-induced changes in leukocyte counts and the enhanced expression of CD11b/CD18 on granulocytes. Importantly, rHDL infusion per se, before LPS administration, was associated with a downregulation of CD14, the main LPS receptor, on monocytes. This effect was biologically relevant, since monocytes isolated from rHDL-treated whole blood showed reduced expression of CD14 and diminished TNF production upon stimulation with LPS. These results suggest that rHDL may inhibit LPS effects in humans in vivo not only by binding and neutralizing LPS but also by reducing CD14 expression on monocytes.

Fibrinolytic response to tumor necrosis factor in healthy subjects.

Tumor necrosis factor (TNF) may be involved in the disturbance of the procoagulant-fibrinolytic balance in septicemia, leading to microvascular thrombosis. To assess the dynamics of the fibrinolytic response to TNF in humans, we performed a crossover saline-controlled study in six healthy men, investigating the effects of a bolus intravenous injection of recombinant human TNF (50 micrograms/m2) on the stimulation and inhibition of plasminogen activation as well as on plasmin activity and inhibition. TNF induced a brief fourfold increase in the overall plasma plasminogen activator (PA) activity peaking after 1 h (p less than 0.0001), which was associated with rises in the antigenic levels of urokinase-type plasminogen activator (p less than 0.0001) and tissue-type plasminogen activator (p less than 0.0001). Plasminogen activator inhibitor type I antigen remained unchanged in the first hour, but showed a rapid eightfold increase thereafter (p less than 0.0001), which coincided with the decrease in PA activity. Generation of plasmin activity in the first hour was signified by an 11-fold rise in D-dimer levels (p less than 0.0001); inhibition of plasmin was reflected by a 36-fold rise in plasmin-alpha 2 antiplasmin complexes (p less than 0.0001), as well as by a transient 16% decrease in alpha 2-antiplasmin activity (p less than 0.01). In conclusion, TNF induced an early activation of the fibrinolytic system becoming maximal in 1 h, with a rapid inhibition thereafter. Earlier observations in the same subjects showed sustained coagulation activation for 6-12 h. The observed disbalance between the procoagulant and fibrinolytic mechanisms after TNF injection confirms the in vivo relevance of the effects of TNF on vascular endothelium in vitro and may explain the tendency towards microvascular thrombosis in septicemia.

Elimination of interleukin 6 attenuates coagulation activation in experimental endotoxemia in chimpanzees.

The role of interleukin 6 (IL-6) in the toxic sequelae of sepsis is controversial. To assess the part of IL-6 in inflammatory responses to endotoxin, we investigated eight chimpanzees after either a bolus intravenous injection of Escherichia coli endotoxin (n = 4; 4 ng/kg) or after the same dose of endotoxin with a simultaneous bolus intravenous injection of an anti-IL-6 mAb (30 mg; n = 4). Anti-IL-6 did not affect the induction of the cytokine network (tumor necrosis factor [TNF], soluble TNF receptors types I and II, and IL-8) by endotoxin, nor did it influence the occurrence of a neutrophilic leukocytosis and neutrophil degranulation, as monitored by the measurement of elastase-alpha 1-antitrypsin complexes. In contrast, anti-IL-6 markedly attenuated endotoxin-induced activation of coagulation, monitored with the plasma levels of the prothrombin fragment F1+2 and thrombin-antithrombin III complexes, whereas activation of fibrinolysis, determined with the plasma concentrations of plasmin-alpha 2-antiplasmin complexes, remained unaltered. We conclude that IL-6 does not have a feedback effect on the release of other cytokines after injection of endotoxin, and that it is not involved in endotoxin-induced neutrophilia or neutrophil degranulation. IL-6 is, however, an important intermediate factor in activation of coagulation in low grade endotoxemia in chimpanzees.

Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees.

Knowledge of the pathogenetic mechanisms responsible for the activation of the coagulation system associated with endotoxemia is important for the development of improved modalities for prevention and treatment. We analyzed the appearance in plasma of TNF, IL-6, and indices of coagulation and fibrinolytic system activation in normal chimpanzees after intravenous infusion of endotoxin. Endotoxin infusion elicited reproducible and dose-dependent elevations in serum TNF and IL-6, as well as marked increases in thrombin generation in vivo as measured by immunoassays for prothrombin activation fragment F1 + 2, thrombin-antithrombin III complexes, and fibrinopeptide A. Activation of the fibrinolytic mechanism was monitored with assays for plasminogen activator activity and plasmin-alpha 2-antiplasmin complexes. To potentially intervene in the molecular pathways elicited by endotoxin, pentoxifylline, an agent that interrupts "immediate early" gene activation by monocytes, or a potent monoclonal antibody that neutralizes tissue factor-mediated initiation of coagulation, were infused shortly before endotoxin. Pentoxifylline markedly inhibited increases in the levels of TNF and IL-6, as well as the effects on coagulation and fibrinolysis. In contrast, the monoclonal antibody to tissue factor completely abrogated the augmentation in thrombin generation, but had no effect on cytokine levels or fibrinolysis. We conclude that the endotoxin-induced activation of coagulation appears to be mediated by the tissue factor-dependent pathway, the fibrinolytic response triggered by endotoxin is not dependent on the generation of thrombin, and that the release of cytokines may be important in mediating the activation of both the coagulation and the fibrinolytic mechanisms in vivo.

Narrow-band imaging compared with conventional colonoscopy for the detection of dysplasia in patients with longstanding ulcerative colitis.

BACKGROUND AND STUDY AIM: Patients with longstanding ulcerative colitis are at increased risk of developing colorectal cancer. Colonoscopic surveillance is advised, but the detection of neoplasia by conventional colonoscopy is difficult. The aim of this study was to compare the accuracy of narrow-band imaging (NBI), a new imaging technique, with standard colonoscopy for the detection of neoplasia in patients with longstanding ulcerative colitis. PATIENTS AND METHODS: This was a prospective, randomized, crossover study of 42 patients with longstanding ulcerative colitis. All participants underwent NBI and conventional colonoscopy with at least 3 weeks between the procedures. Randomization determined the order of the examinations. Targeted biopsies were taken during both procedures; additional random biopsies were taken at conventional colonoscopy only. The number of patients with neoplasia detected by targeted biopsies was used to assess the sensitivity for each technique. RESULTS: With NBI, 52 suspicious lesions were detected in 17 patients, compared with 28 suspicious lesions in 13 patients detected during conventional colonoscopy. Histopathological evaluation of targeted biopsies revealed 11 patients with neoplasia: in four patients the neoplasia was detected by both techniques, in four patients neoplasia was detected only by NBI, and in three patients neoplasia was detected only by conventional colonoscopy ( P = 0.705). Aside from targeted biopsies, 1522 random biopsies were taken. These revealed one additional patient with dysplasia that was not detected by either technique. CONCLUSIONS: The sensitivity of the studied first-generation NBI system for the detection of patients with neoplasia seems to be comparable to conventional colonoscopy, although more suspicious lesions were found during NBI. We believe that it is still too early to stop taking additional random biopsies at surveillance colonoscopy in patients with ulcerative colitis.

A phase II dose ranging, double-blind, placebo-controlled study of alicaforsen enema in subjects with acute exacerbation of mild to moderate left-sided ulcerative colitis.

BACKGROUND: Alicaforsen is an antisense oligonucleotide designed to inhibit expression of human intercellular adhesion molecule 1. Previous clinical studies have demonstrated activity of alicaforsen enema in ulcerative colitis and pouchitis. AIM: To determine the minimally effective dosing regimen of alicaforsen enema in subjects with mild to moderate left-sided ulcerative colitis. METHODS: Randomized, placebo-controlled, double-blind, two-dose ranging multicentre study. One hundred and twelve subjects were equally randomized to receive one of four alicaforsen enema regimens or placebo daily for 6 weeks. Primary end point was Disease Activity Index at week 6. Secondary end points included evaluation of clinical improvement, relapse rates and durability of response. Analysis of data were performed on the intent-to-treat population. RESULTS: No significant difference was observed between treatment arms and placebo in the primary end point. A prolonged reduction in mean% Disease Activity Index relative to baseline was observed in the daily 240 mg alicaforsen enema treatment arm in comparison with placebo from week 18 (51% vs. 18%, P=0.04) to week 30 (50% vs. 11%, P=0.03). CONCLUSIONS: Alicaforsen enema was safe and well tolerated at all doses studied. The durability of the response to alicaforsen enema treatment may suggests a disease-modifying effect.

Maintenance infliximab does not result in increased abscess development in fistulizing Crohn's disease: results from the ACCENT II study.

BACKGROUND: Rapid fistula healing may predispose Crohn's disease patients to abscess development. AIM: Data from ACCENT II were analysed to determine whether fistula-related abscess development is affected by infliximab exposure. METHODS: Following infliximab 5 mg/kg infusions at weeks 0, 2 and 6, patients were evaluated for fistula response for two consecutive visits at least 4 weeks apart. Patients (N = 282) were randomized at week 14 to either placebo or infliximab 5 mg/kg every 8 weeks through week 46. If response was lost at or after week 22, patients could crossover to a 5 mg/kg higher infliximab dose. Fistula-related abscesses were diagnosed by physical examination or by imaging procedures according to usual practice. RESULTS: Infliximab exposure was approximately twofold higher for the infliximab maintenance group. Twenty-one (15%) patients in the infliximab maintenance group had at least one newly developed fistula-related abscess compared with 27 (19%) in the placebo maintenance group (P = 0.526). The proportion of patients with a new fistula-related abscess was similar regardless of whether or not patients crossed over to a 5 mg/kg higher infliximab dose. The number of fistula-related abscesses diagnosed over time did not differ between groups. CONCLUSION: Abscess development in patients with fistulizing Crohn's disease is not dependent on cumulative infliximab exposure.

NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps.

The factors that govern the progression from colonic adenomatous polyp to colon cancer are poorly understood. The observation that NSAIDs act as chemopreventative agents and reduce the size of colonic polyps suggests the involvement of inflammatory signalling, but inflammatory signalling in colonic polyps has not been studied. We investigated the expression of the active forms of NF-kappaB, JNK and p38 MAPK using immunohistochemistry with activation specific antibodies in human colonic adenomas. We show that active NF-kappaB is seen in stromal macrophages that also express COX-2 and TNF-alpha, active JNK is seen in stromal and intraepithelial T-lymphocytes and periendothelial cells of new blood vessels, and active p38 MAPK is most highly expressed in macrophages and other stromal cells. These results demonstrate the presence of active inflammatory signal transduction in colonic polyps and that these are predominantly in the stroma. In the case of NF-kappaB this coincides with the cellular localisation of COX-2. These results support evidence that NSAIDs may act through effects on stromal cells rather than epithelial cells.

Report on the Dutch consensus development meeting for implementation and further development of population screening for colorectal cancer based on FOBT.

A consensus development meeting was held to evaluate whether or not in the Netherlands all requirements were fulfilled for implementation of population screening with FOBT for colorectal cancer, or whether consensus was present that fulfilment by additional research or organisational actions could be obtained within 2-3 years. There was consensus that all classical Wilson and Jungner (1968) criteria, and six additional ones added more recently, had already been fulfilled or could be fulfilled within 2-3 years. Consequently, it was concluded that a national population screening for colorectal cancer should be implemented and carried out in the Netherlands in line with current national and European cancer screening programmes. A list of organisational actions to be taken was established. Research that is needed before the actual national launch of the screening within 2-3 years has been defined. Priorities have to be set for research and organisational actions for the coming 2-3 years for the implementation of population screening. In addition, research suggestions have been defined for the next 10-15 years for evaluation and/or improvement of implemented FOBT screening, and for future screening methodology. It was considered essential that infrastructure for future research would be embedded in the screening programme. A project group to arrange this should be formed.

Procoagulant and proinflammatory activity in acute coronary syndromes.

OBJECTIVES: Both the hemostatic and inflammatory system are thought to play a role in the pathogenesis of acute coronary syndromes. However, their respective contribution and interrelationship remain unclear, therefore, we studied the relationship between activation of the coagulation system and proinflammatory activity in ischemic coronary syndromes. METHODS: Thrombin-antithrombin III (TAT), prothrombin fragments F1 + 2, fibrinopeptide A (FPA), interleukin-6 (IL-6) and interleukin-8 (IL-8) were measured in 50 patients with unstable angina (UA), 60 patients with acute myocardial infarction (AMI) and in 50 patients with stable angina (SA). RESULTS: FPA levels were significantly higher in patients with UA and AMI than in patients with SA (p = 0.0015 and p < 0.0001), and were higher in patients with AMI than UA (p = 0.0013). Plasma IL-6 concentrations were significantly higher in patients with UA and AMI than in patients with SA (p = 0.0020 and p < 0.001), and again were higher in AMI than UA (p = 0.001). Interestingly, FPA or IL-6 elevations on admission were found in different patients. In contrast, TAT, F1 + 2 and IL-8 levels were not different between the three groups. CONCLUSIONS: IL-6 and FPA were shown to be independent predictive markers with equal discriminative power to distinguish stable (SA) from unstable (UA + AMI) patients. Moreover, hemostatic and inflammatory markers can be elevated independently in the acute phase of ischemic coronary syndromes.

Soluble receptors for tumour necrosis factor: a putative marker of disease progression in HIV infection.

OBJECTIVE: To assess the value of concentrations of soluble receptors for tumour necrosis factor (sTNFR) as markers for disease progression in HIV infection. DESIGN: We measured concentrations of sTNFR in the serum of 32 HIV-infected male patients in various stages of disease and in 12 healthy male control subjects. Correlations between the levels of sTNFR and CD4+ lymphocyte counts were calculated. RESULTS: Serum levels of sTNFR p55 and p75 were elevated in parallel with severity of clinical stage. sTNFR p55 levels were higher at later stages of HIV infection (Centers for Disease Control stage IV) with or without concurrent illness, whereas sTNFR p75 was already elevated in asymptomatic carriers, compared with controls. There was an inverse correlation between sTNFR concentrations and CD4+ lymphocyte counts. CONCLUSIONS: Our results suggest that sTNFR concentrations could be potential markers for disease progression in HIV infection.

Diarrhoea in HIV-infected patients: no evidence of cytokine-mediated inflammation in jejunal mucosa.

OBJECTIVE: To determine whether a mucosal cytokine-mediated inflammatory response is involved in cryptosporidial or microsporidial diarrhoea, as well as in diarrhoea of unknown origin in HIV-infected patients. DESIGN: Prospective study. METHODS: Jejunal biopsies were obtained from HIV-infected patients with diarrhoea. Controls were HIV-infected and HIV-seronegative patients without diarrhoea. Two biopsies were homogenized immediately and two other biopsies were first cultured for 20 h. Cytokines [tumour necrosis factor (TNF), interleukin (IL)-1 beta, IL-6, IL-8, IL-10], soluble TNF receptors (sTNFR) p55 and p75, and soluble IL-2 receptor (sIL-2R) were assessed in the homogenates and in the supernatants by sandwich enzyme-linked immunosorbent or enzyme-linked binding assays. The cytokine receptors were also measured in serum. RESULTS: Six HIV-infected patients with cryptosporidiosis, six with microsporidiosis, seven with diarrhoea of unknown origin, seven without diarrhoea, and seven HIV-seronegative patients were eligible. Four patients were excluded because of the presence of other pathogens. No cytokines were detected in immediately homogenized jejunal tissue. Following culture, IL-6 and IL-8 levels were higher in HIV-infected patients with diarrhoea of unknown origin than in HIV-seronegative controls without diarrhoea, although this was not statistically significant. No differences in serum or post-culture supernatant sTNFR p55 and p75 levels existed between the HIV-infected patients with or without diarrhoea. sTNFR, IL-1 beta, IL-10 and the sIL-2R were only detected in low amounts or not at all, and were equally distributed among all patient groups. CONCLUSIONS: This study indicates that mucosal cytokine-mediated inflammatory responses do not play an important role in the pathogenesis of different types of diarrhoea in HIV-infected patients. These results do not support the use of immunomodulatory therapy in these patients.

Lipopolysaccharide recognition, internalisation, signalling and other cellular effects.

Despite the importance of bacterial lipopolysaccharide (LPS) in infection and inflammation, many aspects of LPS action remain poorly understood. Especially, the mechanisms by which cells recognise and react to endotoxins or endotoxin-containing particles and how cellular responses are translated into systemic effects have long remained obscure. However, the recent identification of Toll-like receptors as essential participants in endotoxin signal transduction has provided the first answers in clarifying cellular LPS responses. In this review, we discuss the consequences of the clarification of the cellular effects of LPS. Furthermore, for LPS to exert its effects, it has to be transported to its target cells and be recognised before signalling may be induced, and we shall review the current state of affairs with regard to these recognition processes. Finally, we shall investigate how current knowledge may explain endotoxin neutralisation and subsequent detoxification, either through LPS internalisation or via LPS immobilisation, or through the actions of LPS-binding molecules.

Cyclooxygenase-dependent signalling: molecular events and consequences.

Non-steroidal anti-inflammatory drugs (NSAIDs) currently attract large interest. Next to pain relief, NSAIDs have important anti-thrombotic and anti-oncogenic effects. NSAIDs exert their action by inhibition of cyclooxygenase, the enzyme responsible for the production of prostanoids. Prostanoid signal transduction is still poorly understood, but it has become clear that these inflammatory lipids influence cellular physiology at three different levels: (1) activation of a 7 x transmembrane receptor coupled to heterotrimeric G proteins, (2) the inhibition of inflammation by activating corticosteroid-like receptors, (3) participation in receptor protein tyrosine kinase signal transduction. In this review prostanoid signalling at these three different levels will be reviewed and the relevance in (patho)physiological processes will be evaluated.