Antimicrobial and Anti-inflammatory Gallium-Defensin Surface Coatings for Implantable Devices.

Emerging and re-emerging infections are a global threat driven by the development of antimicrobial resistance due to overuse of antimicrobial agents and poor infection control practices. Implantable devices are particularly susceptible to such infections due to the formation of microbial biofilms. Furthermore, the introduction of implants into the body often results in inflammation and foreign body reactions. The antimicrobial and anti-inflammatory properties of gallium (Ga) have been recognized but not yet utilized effectively to improve implantable device integration. Furthermore, defensin (De, hBD-1) has potent antimicrobial activity in vivo as part of the innate immune system; however, this has not been demonstrated as successfully when used in vitro. Here, we combined Ga and De to impart antimicrobial activity and anti-inflammatory properties to polymer-based implantable devices. We fabricated polylactic acid films, which were modified using Ga implantation and subsequently functionalized with De. Ga-ion implantation increased surface roughness and increased stiffness. Ga implantation and defensin immobilization both independently and synergistically introduced antimicrobial activity to the surfaces, significantly reducing total live bacterial biomass. We demonstrated, for the first time, that the antimicrobial effects of De were unlocked by its surface immobilization. Ga implantation of the surface also resulted in reduced foreign body giant cell formation and expression of proinflammatory cytokine IL-1ß. Cumulatively, the treated surfaces were able to kill bacteria and reduce inflammation in comparison to the untreated control. These innovative surfaces have the potential to prevent biofilm formation without inducing cellular toxicity or inflammation, which is highly desired for implantable device integration.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) enriched mixed micelles (MM): A new nano-drug delivery system with anti-inflammatory potential?

Phosphatidylserine (PS) and phosphatidylglycerol (PG) are naturally occurring phospholipids (PL) with intrinsic anti-inflammatory properties. The therapeutic potential of PS and PG has not been extensively explored and the main focus had been directed towards PS- and PG-liposomes. In order to increase the formulation options, we explored whether mixed micelles (MM) could be an alternative to liposomes. Potential advantages of MM are their thermodynamic stability, small size and ease of manufacture. DOPS- and DOPG-enriched MM were obtained via a co-precipitation technique and physicochemical characterization was performed. The MM, approximately 10 nm in diameter, showed no toxicity on fibroblast cell lines in vitro and virtually no hemolytic activity. The MM suppressed the TNFα-production of mIFNγ/LPS-stimulated mouse peritoneal macrophages (MPM) in vitro similar to DOPS- and DOPG-liposomes. Therefore, DOPS- and DOPG-loaded MM are promising new options for the treatment of inflammatory diseases.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) nanodispersions as potential anti-inflammatory therapeutics: Comparison of in vitro activity and impact of pegylation.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with putative anti-inflammatory potential. However, studies comparing PS and PG are rare and were mainly conducted with phospholipid-dispersions of large size and broad distributions. Thus, we prepared small-sized PS- and PG-loaded liposomes exhibiting narrow distribution, and additionally studied the impact of liposome-pegylation on the reduction of the TNFα-production caused by the PS- and PG-liposomes. These PS- and PG-containing nanodispersions had a small size around 100nm and a narrow distribution (PDI<0.1). The liposome-dispersions showed no toxicity in NHDF- and 3T3-cells and virtually no hemolytic activity. They decreased the TNFα-production of LPS-(lipopolysaccharide)-stimulated mouse peritoneal macrophages in vitro. PG-liposomes always decreased the TNFα-levels more potently than PS-liposomes. Pegylation of PS- and PG-liposomes caused different Zeta potentials, but did not change biological activity. The results of the current study indicate a high potential of the tested formulations for phospholipid-based anti-inflammatory therapies.

The differential statin effect on cytokine production of monocytes or macrophages is mediated by differential geranylgeranylation-dependent Rac1 activation.

Monocytes and macrophages contribute to pathogenesis of various inflammatory diseases, including auto-inflammatory diseases, cancer, sepsis, or atherosclerosis. They do so by production of cytokines, the central regulators of inflammation. Isoprenylation of small G-proteins is involved in regulation of production of some cytokines. Statins possibly affect isoprenylation-dependent cytokine production of monocytes and macrophages differentially. Thus, we compared statin-dependent cytokine production of lipopolysaccharide (LPS)-stimulated freshly isolated human monocytes and macrophages derived from monocytes by overnight differentiation. Stimulated monocytes readily produced tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Statins did not alter cytokine production of LPS-stimulated monocytes. In contrast, monocyte-derived macrophages prepared in the absence of statin lost the capacity to produce cytokines, whereas macrophages prepared in the presence of statin still produced cytokines. The cells expressed indistinguishable nuclear factor-kB activity, suggesting involvement of separate, statin-dependent regulation pathways. The presence of statin was necessary during the differentiation phase of the macrophages, indicating that retainment-of-function rather than costimulation was involved. Reconstitution with mevalonic acid, farnesyl pyrophosphate, or geranylgeranyl pyrophosphate blocked the retainment effect, whereas reconstitution of cholesterol synthesis by squalene did not. Inhibition of geranylgeranylation by GGTI-298, but not inhibition of farnesylation or cholesterol synthesis, mimicked the retainment effect of the statin. Inhibition of Rac1 activation by the Rac1/TIAM1-inhibitor NSC23766 or by Rac1-siRNA (small interfering RNA) blocked the retainment effect. Consistent with this finding, macrophages differentiated in the presence of statin expressed enhanced Rac1-GTP-levels. In line with the above hypothesis that monocytes and macrophages are differentially regulated by statins, the CD14/CD16-, merTK-, CX3CR1-, or CD163-expression (M2-macrophage-related) correlated inversely to the cytokine production. Thus, monocytes and macrophages display differential Rac1-geranylgeranylation-dependent functional capacities, that is, statins sway monocytes and macrophages differentially.

A triple chain polycationic peptide-mimicking amphiphile - efficient DNA-transfer without co-lipids.

Non-viral gene delivery in its current form is largely dependent upon the ability of a delivery vehicle to protect its cargo in the extracellular environment and release it efficiently inside the target cell. Also a simple delivery system is required to simplify a GMP conform production if a marketing authorization is striven for. This work addresses these problems. We have developed a synthetic polycationic peptide-mimicking amphiphile, namely DiTT4, which shows efficient transfection rates and good biocompatibility without the use of a co-lipid in the formulation. The lipid-nucleic acid complex (lipoplex) was characterized at the structural (electron microscopy), physical (laser Doppler velocimetry and atomic force microscopy) and molecular levels (X-ray scattering). Stability studies of the lipoplexes in the presence of serum and heparin indicated a stable formation capable of protecting the cargo against the extracellular milieu. Hemocompatibility studies (hemolysis, complement activation and erythrocyte aggregation) demonstrated the biocompatibility of the formulation for systemic administration. The transfection efficiency was assessed in vitro using the GFP assay and confocal laser scanning microscopy studies. With the chorioallantoic membrane model, an animal replacement model according to the 3R strategy (replacement, refinement, and reduction), initial in vivo experiments were performed which demonstrate fast and efficient transfection in complex tissues and excellent biocompatibility.

Nucleic acid carrier composed of a branched fatty acid lysine conjugate-Interaction studies with blood components.

For the development of gene therapeutics for systemic administration several hurdles have to be overcome. In this article we screen the branched fatty acid lysine conjugate T14diLys, a newly designed cationic lipid for lipofection, regarding this problem. The structure and particle size of lipoplexes, prepared with lipid formulations which are based on these lipid as nucleic acid complexing agent, are investigated in absence and presence of serum. Nuclease digestion assays were performed to evaluate the protective characteristics of the lipid formulation for the complexed nucleic acid. Furthermore, the lipid formulation is investigated regarding the interaction with different serum proteins to get first insights into the protein corona formation. Another focus is set on the hemocompatibility using in vitro assays for hemolysis and complement activation and the irritation test at the chorion allantois membrane of the chicken embryo as in vivo model. Finally, preliminary transfection efficiency studies with cell culture models for cells which are assessable via systemic administration are performed to evaluate possibilities for future therapeutic applications of the new lipid formulations. Summarizing, T14diLys with the co-lipid DOPE can be used to prepare a lipoplex formulation which can be applied systemically and can be used to develop gene therapeutics for targeting endothelial cells, macrophages, or leucocytes.

Prognostic relevance of the interaction between short-term, metronome-paced heart rate variability, and inflammation: results from the population-based CARLA cohort study.

AIMS: To determine the interaction between HRV and inflammation and their association with cardiovascular/all-cause mortality in the general population. METHODS AND RESULTS: Subjects of the CARLA study (n = 1671; 778 women, 893 men, 45-83 years of age) were observed for an average follow-up period of 8.8 years (226 deaths, 70 cardiovascular deaths). Heart rate variability parameters were calculated from 5-min segments of 20-min resting electrocardiograms. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), and soluble tumour necrosis factor-alpha receptor type 1 (sTNF-R1) were measured as inflammation parameters. The HRV parameters determined included the standard deviation of normal-to-normal intervals (SDNN), the root-mean-square of successive normal-interval differences (RMSSD), the low- and high-frequency (HF) power, the ratio of both, and non-linear parameters [Poincaré plot (SD1, SD2, SD1/SD2), short-term detrended fluctuation analysis]. We estimated hazard ratios by using covariate-adjusted Cox regression for cardiovascular and all-cause mortality incorporating an interaction term of HRV/inflammation parameters. Relative excess risk due to interactions (RERIs) were computed. We found an interaction effect of sTNF-R1 with SDNN (RERI: 0.5; 99% confidence interval (CI): 0.1-1.0), and a weaker effect with RMSSD (RERI: 0.4; 99% CI: 0.0-0.9) and HF (RERI: 0.4; 99% CI: 0.0-0.9) with respect to cardiovascular mortality on an additive scale after covariate adjustment. Neither IL-6 nor hsCRP showed a significant interaction with the HRV parameters. CONCLUSION: A change in TNF-α levels or the autonomic nervous system influences the mortality risk through both entities simultaneously. Thus, TNF-α and HRV need to be considered when predicating mortality.

Acidosis differently modulates the inflammatory program in monocytes and macrophages.

Inflammation, ischemia or the microenvironment of solid tumors is often accompanied by a reduction of extracellular pH (acidosis) that stresses the cells and acts on cellular signaling and transcription. The effect of acidosis on the expression of various inflammatory markers, on functional parameters (migration, phagocytic activity) and on signaling pathways involved was studied in monocytic cells and macrophages. In monocytic cell lines acidosis led to a reduction in expression of most of the inflammatory mediators, namely IL-1ß, IL-6, TNF-α, MCP-1, COX-2 and osteopontin. In primary human monocytes MCP-1 and TNF-α were reduced but COX-2 and IL-6 were increased. In RAW264.7 macrophage cell line IL-1ß, COX-2 and iNOS expression was increased, whereas MCP-1 was reduced similar to the effect in monocytic cells. For primary human monocyte-derived macrophages the regulation of inflammatory markers by acidosis depended on activation state, except for the acidosis-induced downregulation of MCP-1 and TNF-α. Acidosis affected functional immune cell behavior when looking at phagocytic activity which was increased in a time-dependent manner, but cellular motility was not changed. Neither ERK1/2 nor CREB signaling was stimulated by the reduction of extracellular pH. However, p38 was activated by acidosis in RAW264.7 cells and this activation was critical for the induction of IL-1ß, COX-2 and iNOS expression. In conclusion, acidosis may impede the recruitment of immune cells, but fosters inflammation when macrophages are present by increasing the level of COX-2 and iNOS and by functionally forcing up the phagocytic activity.

A macrophage/fibroblast co-culture system using a cell migration chamber to study inflammatory effects of biomaterials.

Chronic inflammatory reactions hamper the use of biomaterials after implantation. Thus, the aim of the study was to develop a novel predictive in vitro macrophage/fibroblast co-culture model based on cell migration chambers that allows a timely and locally controlled interaction of both cell types to study the inflammatory responses of biomaterials in vitro. Here, self-assembled monolayers (SAMs) with different wettability and charge properties were used as model biomaterials on which co-cultures were established by use of fence chambers having internal and external compartments. This allowed establishing separated and mixed co-cultures of both cell types before and after removal of the chamber, respectively. The key advantages of this novel co-culture model included not only to establish a timely-resolved study of cytokine release, but also the ability to assess individual macrophage migration in both macrophage mono-cultures and co-cultures. All inflammatory reactions in terms of macrophage adhesion, macrophage migration, foreign body giant cell (FBGC) formation, ß1 integrin expression and pro-inflammatory cytokine production were found strongly surface property dependent. The results show that the hydrophobic CH3 surface caused the strongest inflammatory reactions, whereas the hydrophilic/anionic COOH surface caused the least inflammatory response, indicating low and high biocompatibility of the surfaces, respectively. Most importantly, we found that both macrophage motility and directional movement were increased in the presence of fibroblasts in co-cultures compared with macrophage mono-cultures. Overall, the novel co-culture system provides access to a range of parameters for studying inflammatory reactions and reveals how material surface properties affect the inflammatory responses.

Direct inhibition, but indirect sensitization of pacemaker activity to sympathetic tone by the interaction of endotoxin with HCN-channels.

In critically ill patients regulation of heart-rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization-activated cyclic nucleotide-gated cation-(HCN)-channels may interfere with heart-rate regulation. This study analyzes the effect of LPS, the HCN-channel blocker ivabradine or Ca(2+) -channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart-rate of adult CD1-mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 µg/mL) reduced the beating-rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating-rate, indicating interaction of both compounds with HCN-channels, while coincubation with Ca(2+) -channel blockers and LPS caused additive beating-rate reduction. In CD1-mice (containing an active autonomic-nervous-system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart-rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart-rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN-channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN-channels for sympathetic activation (tachycardic-effect), and in parallel directly inhibits channel activity (bradycardic-effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction.

Interleukin-1 potently contributes to 25-hydroxycholesterol-induced synergistic cytokine production in smooth muscle cell-monocyte interactions.

OBJECTIVES: Inflammation is essential for atherogenesis. Cholesterol, a cardiovascular risk factor, may activate inflammation in the vessel wall during this process. Cytokine-mediated interactions of human monocytes with vascular smooth muscle cells (SMCs) may perpetuate this process. METHODS: We investigated the capacity of the cholesterol metabolite 25-hydroxycholesterol to induce inflammatory mediators in cocultures of freshly isolated monocytes with SMCs. We determined the role of interleukin-(IL)-1 in this interaction using qPCR, bioassays, ELISA and western blot. Cocultures with SMC to monocyte ratios from 1:4 to 1:20 were tested. RESULTS: In separate SMC and monocyte cultures (monocultures) 25-hydroxycholesterol only poorly activated IL-1, IL-6 and MCP-1 production, whereas LPS stimulated much higher cytokine levels than unstimulated cultures. In contrast, cocultures of SMCs and monocytes stimulated with 25-hydroxycholesterol produced hundredfold higher cytokine levels than the corresponding monocultures. Blocking experiments with IL-1-receptor antagonist showed that IL-1 decisively contributed to the 25-hydroxycholesterol-induced synergistic IL-6 and MCP-1 production. The presence of intracellular IL-1ß precursor, released mature IL-1ß, and caspase-1 p10 indicated that the inflammasome was involved in this process. Determination of IL-1-mRNA in Transwell experiments indicated that the monocytes are the major source of IL-1, which subsequently activates the SMCs, the primary source of IL-6 in the coculture. CONCLUSION: Taken together, these interactions between local vessel wall cells and invading monocytes may multiply cholesterol-triggered inflammation in the vessel wall, and IL-1 may play a key role in this process. The data also indicate that lower cholesterol levels than expected from monocultures may suffice to initiate inflammation in the tissue.

Inflammation and renal function after a four-year follow-up in subjects with unimpaired glomerular filtration rate: results from the observational, population-based CARLA cohort.

BACKGROUND: There is evidence that chronic inflammation is associated with the progression/development of chronic renal failure; however, relations in subjects with preserved renal function remain insufficiently understood. OBJECTIVE: To examine the association of inflammation with the development of renal failure in a cohort of the elderly general population. METHODS: After excluding subjects with reduced estimated glomerular filtration rate (eGFR<60 mL/min/1.73 m2) and missing data, the cohort incorporated 785 men and 659 women (aged 45-83 years). Follow-up was performed four years after baseline. Covariate adjusted linear and logistic regression models were used to assess the association of plasma/serum concentrations of soluble tumour necrosis factor receptor 1 (sTNF-R1), C-reactive protein (CRP), and interleukin 6 (IL-6) with change in eGFR/creatinine. The areas under the curve (AUCs) from receiver operating characteristics (ROCs) were estimated. RESULTS: In adjusted models sTNF-R1 was distinctively associated with a decline in eGFR in men (0.6 mL/min/1.73 m2 per 100 pg/mL sTNF-R1; 95% CI: 0.4-0.8), but not in women. A similar association could not be found for CRP or IL-6. Estimates of sTNF-R1 in the cross-sectional analyses were similar between sexes, while CRP and IL-6 were not relevantly associated with eGFR/creatinine. CONCLUSION: In the elderly male general population with preserved renal function sTNF-R1 predicts the development of renal failure.

Inflammation and prolonged QT time: results from the Cardiovascular Disease, Living and Ageing in Halle (CARLA) study.

BACKGROUND: Previous research found an association of CRP with QT time in population based samples. Even more, there is evidence of a substantial involvement of the tumor necrosis factor-alpha system in the pathophysiology of cardiac arrhythmia, while the role of Interleukin 6 remains inconclusive. OBJECTIVE: To determine the association between inflammation with an abnormally prolonged QT-time (APQT) in men and women of the elderly general population. METHODS: Data descend from the baseline examination of the prospective, population-based Cardiovascular Disease, Living and Ageing in Halle (CARLA) Study. After exclusion of subjects with atrial fibrillation and missing ECG recording the final study cohort consisted of 919 men and 797 women. Blood parameters of inflammation were the soluble TNF-Receptor 1 (sTNF-R1), the high-sensitive C-reactive protein (hsCRP), and Interleukin 6 (IL-6). In accordance with major cardiologic societies we defined an APQT above a QT time of 460 ms in women and 450 ms in men. Effect sizes and the corresponding 95% confidence intervals (CI) were estimated by performing multiple linear and logistic regression analyses including the analysis of sex differences by interaction terms. RESULTS: After covariate adjustment we found an odds ratio (OR) of 1.89 (95% CI: 1.13, 3.17) per 1000 pg/mL increase of sTNF-R1 in women, and 0.74 (95% CI: 0.48, 1.15) in men. In the covariate adjusted linear regression sTNF-R1 was again positively associated with QT time in women (5.75 ms per 1000 pg/mL, 95% CI: 1.32, 10.18), but not in men. Taking possible confounders into account IL-6 and hsCRP were not significantly related to APQT in both sexes. CONCLUSION: Our findings from cross-sectional analyses give evidence for an involvement of TNF-alpha in the pathology of APQT in women.

Inhibition of cardiac pacemaker channel hHCN2 depends on intercalation of lipopolysaccharide into channel-containing membrane microdomains.

Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that ß-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

Severity of cardiac impairment in the early stage of community-acquired sepsis determines worse prognosis.

INTRODUCTION: In sepsis, the reduced systemic vascular resistance (SVR) can lead to a compensatory increase in cardiac output (CO). This may mimic a normal cardiac function although there is already a sepsis-induced myocardial depression. On a cohort of patients with septic multi-organ dysfunction syndrome, we have recently developed a method to correlate the actual CO to the afterload (estimated by SVR) and introduced the parameter "afterload-related cardiac performance" (ACP), which indicates if the rise of CO is adequate for the particular SVR. In this present study it was to be investigated, if ACP can reveal septic cardiomyopathy in patients with community-acquired sepsis in the early state soon after admission to the emergency department (ED), and if there is a prognostic relevance of septic cardiomyopathy defined by ACP. Results were compared to cardiac index (CI) and cardiac power index (CPI). METHODS: Adults presenting at the ED with sepsis were included. ACP, CI and CPI were calculated at the time of admission, after 24, and 72 h. They were correlated to severity of disease and the prognostic values were analyzed. RESULTS: A total of 141 patients were included. Only ACP was significantly influenced by severity of sepsis, whereas CI and CPI were not. ACP was the only hemodynamic parameter predicting mortality: nonsurvivors had lower ACP values at the time of admission to the ED (66.9 vs. 88.9 %, p < 0.05) and ACP predicted non-survival with an AUC value of 0.72, p = 0.003. Cardiac impairment defined by an ACP value of 80 % or below determined worse prognosis. CONCLUSIONS: Septic cardiomyopathy occurs already at the early stage of disease and is of prognostic relevance. It might be recognized best, if cardiac function is correlated to afterload.

Interleukin-6, -7, -8 and -10 predict outcome in acute myocardial infarction complicated by cardiogenic shock.

BACKGROUND: The IABP-SHOCK-trial was a morbidity-based randomized controlled trial in patients with infarction-related cardiogenic shock (CS), which used the change of the quantified degree of multiorgan failure as determined by APACHE II score over a 4-day period as primary outcome measure. The prospective hypothesis was that adding IABP therapy to "standard care" would improve CS-triggered multi organ dysfunction syndrome (MODS). The primary endpoint showed no difference between conventionally managed cardiogenic shock patients and those with IABP support. In an inflammatory marker substudy, we analysed the prognostic value of interleukin (IL)-1ß, -6, -7, -8, and -10 in patients with acute myocardial infarction complicated by cardiogenic shock. DESIGN: Inflammatory marker substudy of the prospective, randomized, controlled, open label IABP-SHOCK-trial (ClinicalTrials.gov ID-NCT00469248). SETTING AND METHODS: A single-center study was performed in a 12-bed Intensive-Care-Unit in an university hospital in which 40 consecutive patients were enrolled with an observational period of 96 h. RESULTS: The pro- and anti-inflammatory markers IL-6, -7, -8 and -10 showed a predictive power for mortality of infarct-related CS patients, while IL-1ß did not discriminate. The maximal values during the observational period, in case of IL-7 the minimal value, showed the best power to predict mortality. Both, ROC and multivariate analyses confirmed these suggestions (area under the curve: IL-8, 0.80 ± 0.08; IL-6, 0.79 ± 0.08; IL-10, 0.76 ± 0.08; IL-7, 0.69 ± 0.08). Inflammatory markers were not affected by the presence of IABP support. CONCLUSION: The inflammatory response in patients with myocardial infarction complicated by cardiogenic shock, as reflected by the inflammatory markers IL-6, IL-7, IL-8 and IL-10, demonstrates a clinically relevant prognostic contribution to clinical outcome.

Staphylococcus aureus α-toxin triggers the synthesis of B-cell lymphoma 3 by human platelets.

The frequency and severity of bacteremic infections has increased over the last decade and bacterial endovascular infections (i.e., sepsis or endocarditis) are associated with high morbidity and mortality. Bacteria or secreted bacterial products modulate platelet function and, as a result, affect platelet accumulation at sites of vascular infection and inflammation. However, whether bacterial products regulate synthetic events in platelets is not known. In the present study, we determined if prolonged contact with staphylococcal α-toxin signals platelets to synthesize B-cell lymphoma (Bcl-3), a protein that regulates clot retraction in murine and human platelets. We show that α-toxin induced α(IIb)ß(3)-dependent aggregation (EC(50) 2.98 µg/mL ± 0.64 µg/mL) and, over time, significantly altered platelet morphology and stimulated de novo accumulation of Bcl-3 protein in platelets. Adherence to collagen or fibrinogen also increased the expression of Bcl-3 protein by platelets. α-toxin altered Bcl-3 protein expression patterns in platelets adherent to collagen, but not fibrinogen. Pretreatment of platelets with inhibitors of protein synthesis or the mammalian Target of Rapamycin (mTOR) decreased Bcl-3 protein expression in α-toxin stimulated platelets. In conclusion, Staphylococcusaureus-derived α-toxin, a pore forming exotoxin, exerts immediate (i.e., aggregation) and prolonged (i.e., protein synthesis) responses in platelets, which may contribute to increased thrombotic events associated with gram-positive sepsis or endocarditis.

Differential reduction of HCN channel activity by various types of lipopolysaccharide.

Recently it was shown that lipopolysaccharide (LPS) impairs the pacemaker current in human atrial myocytes. It was speculated that reduced heart rate variability (HRV), typical of patients with severe sepsis, may partially be explained by this impairment. We evaluated the effect of various types of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) expressed in HEK293 cells, and on pacemaker channels in native murine sino-atrial node (SAN) cells, in order to determine the structure of LPS necessary to modulate pacemaker channel function. Application of LPS caused a robust inhibition of hHCN2-mediated current (I(hHCN2)) owing to a negative shift of the voltage dependence of current activation and to a reduced maximal conductance. In addition, kinetics of channel gating were modulated by LPS. Pro-inflammatory LPS-types lacking the O-chain did not reduce I(hHCN2), whereas pro-inflammatory LPS-types containing the O-chain reduced I(hHCN2). On the other hand, a detoxified LPS without inflammatory activity, but containing the O-chain reduced I(hHCN2). Similar observations were made in HEK293 cells expressing hHCN4 and in murine SAN cells. This mechanistic analysis showed the novel finding that the O-chain of LPS is required for reduction of HCN channel activity. In the clinical situation the observed modulation of HCN channels may slow down diastolic depolarization of pacemaker cells and, hence, influence heart rate variability and heart rate.

Contribution of vascular cell-derived cytokines to innate and inflammatory pathways in atherogenesis.

Inflammation is a central element of atherogenesis. Innate pathways contribute to vascular inflammation. However, the initial molecular process(es) starting atherogenesis remain elusive. The various risk factors, represented by particular compounds (activators), may cause altered cellular functions in the endothelium (e.g. vascular endothelial cell activation or -dysfunction), in invading cells (e.g. inflammatory mediator production) or in local vessel wall cells (e.g. inflammatory mediators, migration), thereby triggering the innate inflammatory process. The cellular components of innate immunology include granulocytes, natural killer cells and monocytes. Among the molecular innate constituents are innate molecules, such as the toll-like receptors or innate cytokines. Interleukin-1 (IL-1) and IL-6 are among the innate cytokines. Cytokines are potent activators of a great number of cellular functions relevant to maintain or commove homeostasis of the vessel wall. Within the vessel wall, vascular smooth muscle cells (SMCs) can significantly contribute to the cytokine-dependent inflammatory network by: (i) production of cytokines, (ii) response to cytokines and (iii) cytokine-mediated interaction with invading leucocytes. The cytokines IL-1 and IL-6 are involved in SMC-leucocyte interaction. The IL-6 effects are proposed to be mediated by trans-signalling. Dysregulated cellular functions resulting from dysregulated cytokine production may be the cause of cell accumulation, subsequent low-density lipoprotein accumulation and deposition of extracellular matrix (ECM). The deposition of ECM, increased accumulation of leucocytes and altered levels of inflammatory mediators may constitute an 'innate-immunovascular-memory' resulting in an ever-growing response to anew invasion. Thus, SMC-fostered inflammation, promoted by invading innate cells, may be a potent component for development and acceleration of atherosclerosis.

Statins potently reduce the cytokine-mediated IL-6 release in SMC/MNC cocultures.

Inflammatory pathways are involved in the development of atherosclerosis. Interaction of vessel wall cells and invading monocytes by cytokines may trigger local inflammatory processes. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are standard medications used in cardiovascular diseases. They are thought to have anti-inflammatory capacities, in addition to their lipid-lowering effects. We investigated the anti-inflammatory effect of statins in the cytokine-mediated-interaction-model of human vascular smooth muscle cells (SMC) and human mononuclear cells (MNC). In this atherosclerosis-related inflammatory model LPS (lipopolysaccharide, endotoxin), as well as high mobility group box 1 stimulation resulted in synergistic (i.e. over-additive) IL-6 (interleukin-6) production as measured in ELISA. Recombinant IL-1, tumour necrosis factor-α and IL-6 mediated the synergistic IL-6 production. The standard anti-inflammatory drugs aspirin and indomethacin (Indo) reduced the synergistic IL-6 production by 60%. Simvastatin, atorvastatin, fluvastatin or pravastatin reduced the IL-6 production by 53%, 50%, 64% and 60%, respectively. The inhibition by the statins was dose dependent. Combination of statins with aspirin and/or Indo resulted in complete inhibition of the synergistic IL-6 production. The same inhibitors blocked STAT3 phosphorylation, providing evidence for an autocrine role of IL-6 in the synergism. MNC from volunteers after 5 day aspirin or simvastatin administration showed no decreased IL-6 production, probably due to drug removal during MNC isolation. Taken together, the data show that anti-inflammatory functions (here shown for statins) can be sensitively and reproducibly determined in this novel SMC/MNC coculture model. These data implicate that statins have the capacity to affect atherosclerosis by regulating cytokine-mediated innate inflammatory pathways in the vessel wall.