[Gene polymorphism in intensive care patients. Is the course of disease predetermined?]. / Genpolymorphismen beim Intensivpatienten. Ist der Krankheitsverlauf vorbestimmt?

Molecular biology has revolutionized medicine by increasing our understanding of the pathophysiological mechanisms of disease and the ability to assess genetic risk. Individual differences in disease manifestation and course in intensive care medicine often cannot be explained by known phenotypic risk factors alone. Recent data suggest an association between specific genotypes and the risk of adverse clinical outcomes. This includes inflammatory responses (i.e. TNF-alpha, Il-10), infectious diseases such as pneumonia or meningitis, sepsis, ARDS, as well as the mortality of critically injured patients (polytrauma, severe brain trauma). Continued identification of such allotypes and haplotypes may not only provide insight as to why the response to treatment varies amongst individuals in the intensive care unit, but also may potentially decrease morbidity and mortality through improved risk assessment and the administration of prophylactic therapy.

Role of the complement system in ischaemic heart disease: potential for pharmacological intervention.

The complement system is an innate, cytotoxic host defence system that normally functions to eliminate foreign pathogens. However, considerable evidence suggests that complement plays a key role in the pathophysiology of ischaemic heart disease (IHD). Experimental models of acute myocardial infarction (MI) and autopsy specimens taken from acute MI patients demonstrate that complement is selectively deposited in areas of infarction. Furthermore, inhibition of complement activation or depletion of complement components prior to myocardial reperfusion has been shown to reduce complement-mediated tissue injury in numerous animal models. IHD remains a leading cause of patient morbidity and mortality. Considerable effort in recent years has therefore been directed by biotechnology and pharmaceutical industries towards the development of novel, human complement inhibitors. Proposed anticomplement therapeutic strategies include the administration of naturally occurring or recombinant complement regulators, anticomplement monoclonal antibodies, and anticomplement receptor antagonists. Although data regarding the effectiveness of anticomplement therapy in humans is limited at present, a number of novel anticomplement therapeutic strategies are currently in clinical trials. The role of complement in IHD and potential for pharmacological intervention is reviewed.

Endothelial oxidative stress activates the lectin complement pathway: role of cytokeratin 1.

Oxidative stress increases endothelial mannose-binding lectin (MBL) binding and activates the lectin complement pathway (LCP). However, the molecular mechanism of MBL binding to the endothelium after oxidative stress is unknown. Intermediate filaments have been previously reported to activate the classical complement pathway in an antibody-independent manner. We investigated whether oxidative stress increases human umbilical vein endothelial cell (HUVEC) cytokeratin 1 (CK1) expression and activates the LCP via MBL binding to CK1. Reoxygenation (3 hours, 21% O(2)) of hypoxic HUVECs (24 hours, 1% O(2)) significantly increased CK1 mRNA (in situ hybridization) and membrane protein expression [enzyme-linked immunosorbent assay (ELISA)/confocal microscopy]. Incubating human serum (HS) with N-acetyl-D-glucosamine or anti-human MBL monoclonal antibody attenuated MBL and C3 deposition on purified CK1 (ELISA). CK1 and MBL were co-immunoprecipitated from hypoxic HUVECs reoxygenated in HS. Treatment with anti-human cytokeratin Fab fragments attenuated endothelial MBL and C3 deposition after oxidative stress (ELISA/confocal microscopy). We conclude that: 1) endothelial oxidative stress increases CK1 expression, MBL binding, and C3 deposition; 2) inhibition of MBL attenuates purified CK1-induced complement activation; and 3) anti-human cytokeratin Fab fragments attenuate endothelial MBL and C3 deposition after oxidative stress. These results suggest that MBL binding to endothelial cytokeratins may mediate LCP activation after oxidative stress.

The safety of intraoperative transesophageal echocardiography: a case series of 7200 cardiac surgical patients.

UNLABELLED: Transesophageal echocardiography (TEE) is an invaluable intraoperative diagnostic monitor that is considered to be relatively safe and noninvasive. Insertion and manipulation of the TEE probe, however, may cause oropharyngeal, esophageal, or gastric trauma. We report the incidence of intraoperative TEE-associated complications in a single-center series of 7200 adult cardiac surgical patients. Information related to intraoperative TEE-associated complications was obtained retrospectively from the intraoperative TEE data form, routine postoperative visits, and cardiac surgical morbidity and mortality data. The overall incidences of TEE-associated morbidity and mortality in the study population were 0.2% and 0%, respectively. The most common TEE-associated complication was severe odynophagia, which occurred in 0.1% of the study population. Other complications included dental injury (0.03%), endotracheal tube malpositioning (0.03%), upper gastrointestinal hemorrhage (0.03%), and esophageal perforation (0.01%). TEE probe insertion was unsuccessful or contraindicated in 0.18% and 0.5% of the study population, respectively. These data suggest that intraoperative TEE is a relatively safe diagnostic monitor for the management of cardiac surgical patients. IMPLICATIONS: The overall morbidity (0.2%) and mortality (0%) rates of intraoperative transesophageal echocardiography (TEE) were determined in a retrospective case series of 7200 adult, anesthetized cardiac surgical patients. The most common source of TEE-associated morbidity was odynophagia (0.1%), which resolved with conservative management. These results suggest that TEE is a safe diagnostic tool for the management of cardiac surgical patients.

A keratin peptide inhibits mannose-binding lectin.

Complement plays a significant role in mediating endothelial injury following oxidative stress. We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-D-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (K(D)) of 5 x 10(-5) mol/L. Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10-50 microg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation.

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Complement activation after oxidative stress: role of the lectin complement pathway.

The complement system plays an important role in mediating tissue injury after oxidative stress. The role of mannose-binding lectin (MBL) and the lectin complement pathway (LCP) in mediating complement activation after endothelial oxidative stress was investigated. iC3b deposition on hypoxic (24 hours; 1% O(2))/reoxygenated (3 hours; 21% O(2)) human endothelial cells was attenuated by N-acetyl-D-glucosamine or D-mannose, but not L-mannose, in a dose-dependent manner. Endothelial iC3b deposition after oxidative stress was also attenuated in MBL-deficient serum. Novel, functionally inhibitory, anti-human MBL monoclonal antibodies attenuated MBL-dependent C3 deposition on mannan-coated plates in a dose-dependent manner. Treatment of human serum with anti-MBL monoclonal antibodies inhibited MBL and C3 deposition after endothelial oxidative stress. Consistent with our in vitro findings, C3 and MBL immunostaining throughout the ischemic area at risk increased during rat myocardial reperfusion in vivo. These data suggest that the LCP mediates complement activation after tissue oxidative stress. Inhibition of MBL may represent a novel therapeutic strategy for ischemia/reperfusion injury and other complement-mediated disease states.

Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response that causes substantial clinical morbidity. Activation of complement during CPB contributes significantly to this inflammatory process. We examined the capability of a novel therapeutic complement inhibitor to prevent pathological complement activation and tissue injury in patients undergoing CPB. METHODS AND RESULTS: A humanized, recombinant, single-chain antibody specific for human C5, h5G1.1-scFv, was intravenously administered in 1 of 4 doses ranging from 0.2 to 2.0 mg/kg before CPB. h5G1.1-scFv was found to be safe and well tolerated. Pharmacokinetic analysis revealed a sustained half-life from 7.0 to 14.5 hours. Pharmacodynamic analysis demonstrated significant dose-dependent inhibition of complement hemolytic activity for up to 14 hours at 2 mg/kg. The generation of proinflammatory complement byproducts (sC5b-9) was effectively inhibited in a dose-dependent fashion. Leukocyte activation, as measured by surface expression of CD11b, was reduced (P<0.05) in patients who received 1 and 2 mg/kg. There was a 40% reduction in myocardial injury (creatine kinase-MB release, P=0.05) in patients who received 2 mg/kg. Sequential Mini-Mental State Examinations (MMSE) demonstrated an 80% reduction in new cognitive deficits (P<0.05) in patients treated with 2 mg/kg. Finally, there was a 1-U reduction in postoperative blood loss (P<0. 05) in patients who received 1 or 2 mg/kg. CONCLUSIONS: A single-chain antibody specific for human C5 is a safe and effective inhibitor of pathological complement activation in patients undergoing CPB. In addition to significantly reducing sC5b-9 formation and leukocyte CD11b expression, C5 inhibition significantly attenuates postoperative myocardial injury, cognitive deficits, and blood loss. These data suggest that C5 inhibition may represent a novel therapeutic strategy for preventing complement-mediated inflammation and tissue injury.

Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues.

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Effect of sialyl Lewis(x) oligosaccharide on myocardial and cerebral injury in the pig.

BACKGROUND: Although administration of the sialyl Lewis(x) oligosaccharide may reduce myocardial injury after ischemia-reperfusion, its effect on coronary and cerebral microvascular regulation and its clinical application during cardiac operation have not been examined. METHODS: Pigs were placed on normothermic cardiopulmonary bypass after 30 minutes of left anterior descending coronary artery occlusion. The hearts were then arrested with cold high potassium cardioplegia. After 1 hour the cross-clamp was removed and the pigs were weaned from cardiopulmonary bypass and perfused for an additional 1 hour. CY-1503 (a sodium salt of the sialyl Lewis(x) oligosaccharide, n = 6) was administered before reperfusion. Six other pigs received saline vehicle. Endothelium-dependent relaxation of precontracted coronary and brain arterioles (70 to 180 microm) to adenosine 5'-diphosphate and endothelium-independent relaxation to sodium nitroprusside were studied in vitro with videomicroscopy. Control values were obtained from uninstrumented pigs. Myeloperoxidase activity in the myocardium and brain was measured to quantify neutrophil infiltration. Cardiac function and perfusion were assessed by left ventricular systolic pressure, maximum rate of increase of left ventricular pressure, left anterior descending coronary artery blood flow and percent segmental shortening, and cerebral vascular resistance, internal carotid artery blood flow, and the constitutively expressed and inducible isoform of nitric oxide synthase mRNA were measured. RESULTS: The impaired myocardial contractile function after ischemia and cardioplegia was not improved by administration of CY-1503. The reduced endothelium-dependent relaxation responses of coronary and brain arterioles during ischemia followed by cardioplegia and cardiopulmonary bypass were improved with CY-1503, but the altered pattern of organ perfusion was not improved. Myeloperoxidase activity was increased in the heart after ischemia-cardioplegia and in the brain after cardiopulmonary bypass. CY-1503 reduced myeloperoxidase activity in both the myocardium and in the brain. Expressions of myocardial inducible isoform or constitutively expressed nitric oxide synthase were not altered in the heart. CONCLUSIONS: Although the sialyl Lewis(x) oligosaccharide does reduce neutrophil infiltration and endothelial injury in the coronary and cerebral microcirculation after cardiopulmonary bypass, it does not have significant beneficial acute effects on organ perfusion or function in the myocardium or brain.

Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells.

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 +/- 0. 6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-alpha also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

Complement activation following oxidative stress.

It is clear that complement plays an important role in the inflammatory process following oxidative stress in cellular and animal models. Clinical trials underway with novel complement inhibitors will establish the potential therapeutic benefit of complement inhibition in human disease. For as much as we understand about the role of complement in disease states, many questions remain. How is complement activated on endothelial cells following oxidative stress? What is the ligand for MBL on endothelial cells following oxidative stress? Will inhibition of MBL provide tissue protection to the extent observed with other complement inhibitors such as sCR1 or anti-C5 mAbs? These questions and more will undoubtedly be answered in the next millennium.

Complement activation following reoxygenation of hypoxic human endothelial cells: role of intracellular reactive oxygen species, NF-kappaB and new protein synthesis.

Complement plays an important role in ischemia-reperfusion injury. We recently demonstrated that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) activated the classical complement pathway and augmented iC3b deposition. In the present study, we investigated the potential role of oxygen-derived free radicals, NF-kappaB and new protein synthesis in this model. HUVECs subjected to 12 or 24 h hypoxic stress (1% O2) and then reoxygenated (0.5, 1, 2 or 3 h; 21% O2) in 30% human serum activated complement and deposited iC3b. Addition of hydrogen peroxide (H2O2; 1-100 micromol/l) to normoxic HUVECs increased iC3b deposition in a concentration-dependent manner. H2O2 (10 micromol/l), a concentration that did not significantly increase iC3b deposition on normoxic HUVECs, augmented iC3b deposition on hypoxic/reoxygenated HUVECs. We observed a significant increase in intracellular H2O2 and hydroxyl radical (OH.) production in hypoxic/reoxygenated HUVECs using dihydrorhodamine 123. Further, treatment of HUVECs with dimethylthiourea (DMTU, 1-100 micromol/l), deferoxamine (DEF, 1-100 micromol/l), or oxypurinol (10 micromol/l), but not superoxide dismutase (SOD, 500 U/ml), catalase (300 U/ml) or iron-loaded DEF, attenuated iC3b deposition following hypoxia/reoxygenation in a concentration-dependent manner. Western analysis demonstrated hypoxia-induced nuclear NF-kappaB translocation that increased with reoxygenation. Inhibition of new protein synthesis (i.e. cycloheximide) or inhibition of NF-kappaB (ALLN or SN-50) also significantly decreased iC3b deposition on hypoxic/reoxygenated HUVECs. We conclude that (1) hypoxic/reoxygenated HUVECs generate H2O2 and OH.; (2) treatment of HUVECs with cell permeable reactive oxygen species inhibitors/scavengers (i.e. DEF, DMTU, oxypurinol) but not large molecular weight inhibitors (i.e. catalase or SOD) significantly reduces iC3b deposition and (3) inhibition of new protein synthesis or NF-kappaB activation attenuates iC3b deposition. These data suggest that iC3b deposition on the vascular endothelium may be regulated by intracellular oxygen-derived free radical-induced activation of NF-kappaB, new protein synthesis and activation of the classical complement pathway during ischemia/reperfusion.