Phagocytosis of live and dead Escherichia coli and Staphylococcus aureus in human whole blood is markedly reduced by combined inhibition of C5aR1 and CD14.

BACKGROUND: Sepsis is a dysregulated host response to infection. The aim of this study was to investigate the effects of complement- and CD14 inhibition on phagocytosis of live and dead Gram-negative and Gram-positive bacteria in human whole blood. METHODS: Lepirudin-anticoagulated blood was incubated with live or dead E. coli or S. aureus at 37 °C for 120 min with or without the C5aR1 antagonist PMX53 and/or anti-CD14. Granulocyte and monocyte phagocytosis were measured by flow cytometry, and five plasma cytokines by multiplex, yielding a total of 28 mediators of inflammation tested for. RESULTS: 16/28 conditions were reduced by PMX53, 7/28 by anti-CD14, and 24/28 by combined PMX53 and CD14 inhibition. The effect of complement inhibition was quantitatively more pronounced, in particular for the responses to S. aureus. The effect of anti-CD14 was modest, except for a marked reduction in INF-ß. The responses to live and dead S. aureus were equally inhibited, whereas the responses to live E. coli were inhibited less than those to dead E. coli. CONCLUSION: C5aR1 inhibited phagocytosis-induced inflammation by live and dead E. coli and S. aureus. CD14 blockade potentiated the effect of C5aR1 blockade, thus attenuating inflammation.

Staphylococcus aureus-induced complement activation promotes tissue factor-mediated coagulation.

Essentials Complement, Toll-like receptors and coagulation cross-talk in the process of thromboinflammation. This is explored in a unique human whole-blood model of S. aureus bacteremia. Coagulation is here shown as a downstream event of C5a-induced tissue factor (TF) production. Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation. SUMMARY: Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.

C1-inhibitor efficiently delays clot development in normal human whole blood and inhibits Escherichia coli-induced coagulation measured by thromboelastometry.

INTRODUCTION: C1-inhibitor (C1-INH), a serine protease inhibitor in plasma plays a central role in the cross-talk among the complement, coagulation, fibrinolytic and kallikrein-kinin systems. However, previous reports indicate thrombotic risks in children following supraphysiological dosing with C1-INH. OBJECTIVE: To investigate the role of supraphysiological C1-INH concentrations in clot development with and without addition of Escherichia coli (E. coli) in fresh human whole blood using thromboelastometry. MATERIALS AND METHODS: Blood was collected in citrate tubes, and C1-INH (3.0 to 47.6µM) or human serum albumin (HSA) was added as a control. Activated partial thromboplastin time (aPTT) was analysed in the plasma. The analyses non-activated thromboelastometry (NATEM), extrinsic (EXTEM) or intrinsic thromboelastometry (INTEM) were performed using rotational thromboelastometry. RESULTS: C1-INH increased aPTT 1.8-fold (p< 0.05), whereas HSA had no effect. C1-INH increased NATEM clotting time (CT) from 789s to 2025 s (p< 0.05) in a dose-dependent manner. C1-INH reduced the NATEM alpha angle from 47 to 28° (p<0.05) and increased the NATEM clot formation time from 261s to 595s (p< 0.05). E. coli significantly reduced the NATEM CT after 120min of incubation. C1-INH prevented E. coli-induced activation (p< 0.05). C1-INH significantly increased the INTEM CT (p< 0.05), but had no effect on EXTEM CT. C1-INH (47.6µM) significantly reduced fibrinolysis measured as NATEM and EXTEM lysis indices LI60. CONCLUSIONS: Supraphysiological C1-INH concentrations have dose-dependent anticoagulant effects in human whole blood in vitro. At very high levels C1-INH also inhibits fibrinolysis.

The key roles of complement and tissue factor in Escherichia coli-induced coagulation in human whole blood.

The complement system and the Toll-like (TLR) co-receptor CD14 play important roles in innate immunity and sepsis. Tissue factor (TF) is a key initiating component in intravascular coagulation in sepsis, and long pentraxin 3 (PTX3) enhances the lipopolysaccharide (LPS)-induced transcription of TF. The aim of this study was to study the mechanism by which complement and CD14 affects LPS- and Escherichia coli (E. coli)-induced coagulation in human blood. Fresh whole blood was anti-coagulated with lepirudin, and incubated with ultra-purified LPS (100 ng/ml) or with E. coli (1 × 10(7) /ml). Inhibitors and controls included the C3 blocking peptide compstatin, an anti-CD14 F(ab')2 antibody and a control F(ab')2 . TF mRNA was measured using quantitative polymerase chain reaction (qPCR) and monocyte TF surface expression by flow cytometry. TF functional activity in plasma microparticles was measured using an amidolytic assay. Prothrombin fragment F 1+2 (PTF1.2) and PTX3 were measured by enzyme-linked immunosorbent assay (ELISA). The effect of TF was examined using an anti-TF blocking antibody. E. coli increased plasma PTF1.2 and PTX3 levels markedly. This increase was reduced by 84->99% with compstatin, 55-97% with anti-CD14 and > 99% with combined inhibition (P < 0·05 for all). The combined inhibition was significantly (P < 0·05) more efficient than compstatin and anti-CD14 alone. The LPS- and E. coli-induced TF mRNA levels, monocyte TF surface expression and TF functional activity were reduced by > 99% (P < 0·05) with combined C3 and CD14 inhibition. LPS- and E. coli-induced PTF1.2 was reduced by 76-81% (P < 0·05) with anti-TF antibody. LPS and E. coli activated the coagulation system by a complement- and CD14-dependent up-regulation of TF, leading subsequently to prothrombin activation.

C1-inhibitor efficiently inhibits Escherichia coli-induced tissue factor mRNA up-regulation, monocyte tissue factor expression and coagulation activation in human whole blood.

Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses.

The effects of selective complement and CD14 inhibition on the E. coli-induced tissue factor mRNA upregulation, monocyte tissue factor expression, and tissue factor functional activity in human whole blood.

BACKGROUND: The complement pathway and CD14 play essential roles in inflammation, but little is known about the relative roles of complement and CD14 in E. coli-induced tissue factor (TF) mRNA upregulation, expression by monocytes, and functional activity in human whole blood. METHODS: Whole E. coli bacteria were incubated for up to 4 h in human whole blood containing the anticoagulant lepirudin, which does not affect complement activation. TF mRNA levels were analyzed using reverse transcription, quantitative real-time PCR (RT-qPCR), and the expression of TF on the cell surface was analyzed using flow cytometry. Complement was selectively inhibited using the C3 convertase inhibitor compstatin or a C5a receptor antagonist (C5aRa), while CD14 was blocked by an anti-CD14 F(ab')2 monoclonal antibody. RESULTS: The E. coli-induced TF mRNA upregulation was reduced to virtually background levels by compstatin, whereas anti-CD14 had no effect. Monocyte TF expression and TF activity in plasma microparticles were significantly reduced by C5aRa. Anti-CD14 alone only slightly reduced E. coli-induced monocyte TF expression but showed a modest additive effect when combined with the complement inhibitors. Inhibiting complement and CD14 efficiently reduced the expression of the E. coli-induced cytokines IL-1beta, IL-6, IL-8, and platelet-derived growth factor bb. CONCLUSION: Our results indicate that E. coli-induced TF mRNA upregulation is mainly dependent on complement activation, while CDI4 plays a modest role in monocyte TF expression and the plasma TF activity in human whole blood.

A young man with acute dilated cardiomyopathy associated with methylphenidate.

An 18-year-old obese man with a body mass index of 40, diagnosed with attention-deficit hyperactivity disorder and treated with methylphenidate (Concerta) was acutely admitted to hospital with hypoxia and dyspnoea. On investigation signs of liver-, renal-, and heart-failure were found. Noradrenalin infusion was started. Echocardiography showed dilated left ventricle and an ejection fraction (EF) of 25%. Liver function improved, noradrenalin and dobutamine were tapered, but three days after admission a new echocardiography showed an EF of 10%. The patient was transferred to the National Hospital (Rikshospitalet, Oslo), where intensified treatment including intra aortic balloon pump (IABP) was instituted. Cardiac function improved, and 3 weeks later the IABP was disconnected. EF at this point was 15%. The patient was denied heart transplantation due to various cofactors. The investigation concluded with a probable relationship between his cardiomyopathy and the use of methylphenidate (Concerta).

Tryptase levels after suxamethonium administration and defibrillation.

BACKGROUND: A more than threefold increase in tryptase, when comparing with the control sample, strengthens the diagnosis of anaphylaxis. Trauma, coronary ischaemia and non-IgE-mediated reactions to several medications have been shown to cause more than threefold rise in tryptase levels. The aim of our study was to examine whether suxamethonium or defibrillation could lead to a more than threefold increase in tryptase in the absence of signs of anaphylaxis. METHODS: S-tryptase was measured in 50 patients who had general anaesthesia with either pentothal and suxamethonium before electro convulsive therapy (ECT) to treat depression (n=31) or propofol before electro conversion to treat atrial fibrillation (n=19). Blood samples were collected minutes before and 1 h after the procedures. RESULTS: Tryptase values did not differ significantly before and after the procedures. CONCLUSION: Tryptase levels do not increase in patients undergoing elective defibrillation or ECT with the administration of suxamethonium, in absence of symptoms of anaphylaxis.

New biomarkers in an acute model of live Escherichia coli-induced sepsis in pigs.

We developed a live Escherichia coli model of acute sepsis in pigs with emphasize on biomarkers reflecting the early inflammatory response of sepsis. Healthy pigs, 25-35 kg, were challenged intravenously (IV) (n = 12) or intrapulmonary (n = 6) with live E. coli and observed for 3 and 5 h respectively. Control pigs received culture medium (n = 6 + 3). Haemodynamic parameters and a broad panel of inflammatory mediators were measured. The dose of bacteria was carefully titrated to obtain a condition resembling the early phase of human septic shock. The IV group displayed a pro-inflammatory response [significant increase in tumour necrosis factor-alpha, interleukin (IL)-6 and IL-8] and an early anti-inflammatory response (significant increase in IL-10). For the first time, we demonstrate a significant increase in IL-12 and matrix metalloproteinase-9 (MMP) early in pig sepsis. Coagulation was activated (significant increase in thrombin-antithrombin complexes) and there was a significant decrease in the serum proteins suggesting capillary leakage. Haemodynamic parameters reflected a septic condition with significant decrease in systemic blood pressure, increases in heart rate, pulmonary artery pressure and base deficit. None of these changes was observed in the control group. Interleukin-1beta and vascular endothelial growth factor increased in both groups. Nitric oxide measurements suggested an initial pulmonary vascular endothelial inflammatory response. The intrapulmonary group, which did not resemble septic condition, showed a substantial increase in MMP-9. In this porcine model of sepsis, IL-12 and MMP-9 were detected for the first time. These biomarkers may have an impact in the understanding and future treatment of sepsis.

Angioedema from angiotensin-converting enzyme (ACE) inhibitor treated with complement 1 (C1) inhibitor concentrate.

BACKGROUND: Up to seven in every 1000 patients experience angioedema from angiotensin-converting enzyme (ACE) inhibitors, even after many years of use. In 2003, every 20th Norwegian used an ACE inhibitor. CASE REPORT: A 61-year-old woman with chronic obstructive pulmonary disease and a past acute myocardial infarction had used 7.5 mg of ramipril daily for the past 7 years. She also used acetylsalicylic acid, simvastatin, theophylline and salmeterol. One night she woke up with edema of the tongue. On hospital arrival, 250 mg of hydrocortisone and 5 mg of dexchlorpheniramine were given intravenously (i.v.) and 0.3 mg of epinephrine was given subcutaneously (s.c.). The edema of the tongue progressed over the next 8 h and made the tongue protrude. Fiberscopy revealed glassy edema of the arytenoids. Inspiratory stridor was heard and the patient could not speak. She became increasingly uneasy and restless. Berinert complement 1 (C1) inhibitor concentrate (1500 units) was administered i.v. Over the following 20 min, stridor gradually subsided, the patient calmed and she was able to talk. DISCUSSION: ACE inhibitor-provoked angioedema shares many clinical features with hereditary angioedema (HAE), including a limited effect of steroids, antihistamines and epinephrine. HAE, caused by excess bradykinin formation as a result of C1 inhibitor deficiency, usually has its laryngeal edema effectively reversed by C1 inhibitor in less than 0.5 h. Although patients experiencing ACE inhibitor-provoked angioedema have normal C1 inhibitor values, as in our patient, excess bradykinin is probably important as ACE breaks down bradykinin. It is unknown why ACE inhibitor-provoked angioedema appears in some and sometimes after many years of use. CONCLUSION: We believe that C1 inhibitor was effective in reversing the ACE inhibitor-induced angioedema in our patient.

Hypotensive shock and angio-oedema from angiotensin II receptor blocker: a class effect in spite of tripled tryptase values.

In adverse reactions with shock, tripled tryptase values can support a diagnosis of anaphylaxis. A 51-year old physically fit woman experienced angio-oedema and hypotensive shock after irbesartan ingestion requiring noradrenaline infusion. Serum tryptase rose to three times the normal value. Total immunoglobulin E and skin prick tests were normal, however. As nonallergic increases in tryptase have been observed, e.g. during angio-oedema from angiotensin-converting enzyme inhibitors, and bradykinin itself can degranulate mast cells acutely, we interpret the reaction as a class effect. To our knowledge, our report is one of the first on shock and angio-oedema from irbesartan.

Neuroleptic malignant syndrome with severe liver failure.

A schizophrenic patient on long-time neuroleptic medication was admitted with ileus. Secondarily, a high fever, rigidity, mental confusion, tachycardia and hypotension developed. After bromocriptine was given, the temperature dropped by 2 degrees C and the patient improved markedly. A diagnosis of neuroleptic malignant syndrome was made. Five years later she was re-admitted with similar symptoms and also severe liver failure. Meanwhile the discontinued neuroleptic medication had been reinstituted. Again bromocriptine reduced the temperature of approximately 2 degrees C, and was paralleled by a normalization of liver function. To our knowledge this is the second report on severe liver failure in conjunction with neuroleptic malignant syndrome. The efficacy of bromocriptine in the treatment of this syndrome is underlined.

C1-inhibitor reduces the ischaemia-reperfusion injury of skeletal muscles in mice after aortic cross-clamping.

BACKGROUND: Both C1-inhibitor (C1-INH) and antibodies against the CD18 adhesion molecule have been shown to reduce ischaemia-reperfusion injuries. The objective of this study was to investigate the effect of increased ischaemia times and to determine whether inhibiting C1 or blocking the CD18 function was protective in skeletal muscle ischaemia-reperfusion injury after aortic cross-clamping. MATERIALS AND METHODS: BALB/c mice were subjected to aortic cross-clamping below the renal artery for 60, 75 or 105 min, followed by 3 h of reperfusion. Two-thirds of a total dose of anti-CD18 antibody (40 mg/kg) or human C1-INH (1,000 IU/kg) was given by intraperitoneal injection before ischaemia and one-third immediately after the clamping. Creatine kinase (CK) in the plasma was used as an indicator of muscle injury severity. RESULTS: There was a consistent rise in the plasma CK concentration proportional to the length of ischaemia (P < 0.0005). C1-INH treatment significantly (P = 0.012) reduced the plasma CK for the ischaemia times of 75 and 105 min. The anti-CD18 antibody did not have any effect, as demonstrated by the CK values that were similar to controls (P = 0.836). CONCLUSION: The data support a beneficial role for C1-INH in the treatment of ischaemia-reperfusion injuries of skeletal muscles.

When is an anesthesiologist needed in a helicopter emergency medical service in northern Norway?

BACKGROUND: A national air ambulance service, including helicopters and airplanes, was implemented in Norway in 1988. The main intention was to offer advanced medical services when needed. All helicopters are manned by anesthesiologists. Catchment areas for the 11 helicopters span from cities to scarcely populated areas, particularly in the north. Our aim was to assess what proportion of ambulance missions carried out by the rescue helicopter in Bodø, northern Norway, delivered advanced medical treatment needing the skills of an anesthesiologist. METHODS: Flight and ambulance records (n = 2078) from 1988 and 1990-98 (10 years) were analyzed retrospectively. Inter-hospital transfers (n = 147) and search- and rescue missions (n = 332) were not included. According to the level of medical treatment given missions were categorized into three groups (A, B and C). Treatment in groups A and B would not require an anesthesiologist. RESULTS: Two thousand and seventy-eight ambulance missions carried 2166 patients (114 per 100 000 per year). Median take-off and on-scene times were 29 and 55 min, respectively. Seven hundred and fifty-five patients (35%) suffered from cardiovascular disease, 495 (23%) were injured and 250 (12%) were parturients. One hundred and seven patients (5.0%) received advanced prehospital emergency treatment requiring an anesthesiologist. Forty-five of the 107 patients survived to discharge from hospital, amongst whom 28 had received intravenous nitroglycerin for angina or suspected myocardial infarction. CONCLUSION: In our rural area, with a widely scattered population, 95% of patients received medical treatment not requiring an anesthesiologist. A selective use of the anesthesiologist seems indicated.

Expression of active human C1 inhibitor serpin domain in Escherichia coli.

Human C1 inhibitor is a highly glycosylated serine protease inhibitor of the serpin family. The protein contains two disulfide bonds. In this study, an N-terminally truncated form of recombinant C1 inhibitor was overexpressed in Escherichia coli strains BL21(DE3) and AD494(DE3), the latter enabling the formation of disulfide bonds within the cytoplasm. With both strains, a major fraction of the recombinant protein produced appeared to be insoluble. However, the soluble fraction of lysates from strain AD494(DE3) inhibited the C1s target protease in functional assays. Recombinant C1 inhibitor produced in this strain also displayed the ability to complex with C1s in vitro. In contrast, lysates from strain BL21(DE3) displayed no C1 inhibitor activity. These data support the notion that glycosylation is not important, whereas disulfide bond formation appears to be essential for the production of an active recombinant C1 inhibitor. Thus, bacterial strains that permit the formation of disulfide bonds may represent a reliable system for the production of recombinant C1 inhibitor. However, a major obstacle to large-scale production will be to produce the protein in a soluble form. Attempts to increase the yield of soluble protein by coexpression of the GroEL/ES chaperonins resulted in an increase in solubility.

[Coronary angiography in a county hospital]. / Koronarangiografi ved et sentralsykehus.

BACKGROUND: At a county hospital in northern Norway, coronary angiographies have been performed for more than 25 years. We wanted to compare our complication rates with results from larger hospitals. MATERIAL AND METHODS: Data concerning indication for angiography, findings and complications were collected retrospectively from the files of 837 patients who underwent coronary angiography at our hospital during the last ten years. The number of procedures per year has varied from 30 to 125. RESULTS: 50% of the patients had three-vessel and/or left main coronary artery stenosis. Less than 10% of the patients had no significant coronary stenosis or valvular disease at angiography. The total complication rate was 4.8%. Nine patients (1.1%) had myocardial infarction or stroke with sequelae; three patients died (0.4%). INTERPRETATION: Our complication rates are higher than those from other larger hospitals in Norway and internationally, but they have decreased over time. The high percentage of serious coronary pathology among our patients may have contributed significantly to the complication rate. In addition, the annual number of exams may have been too low to give sufficient operator training. The results require a continuing evaluation of our practice.

[Coronary angiography in a county hospital]. / Koronarangiografi ved et sentralsykehus.

BACKGROUND: At a county hospital in northern Norway, coronary angiographies have been performed for more than 25 years. We wanted to compare our complication rates with results from larger hospitals. MATERIAL AND METHODS: Data concerning indication for angiography, findings and complications were collected retrospectively from the files of 837 patients who underwent coronary angiography at our hospital during the last ten years. The number of procedures per year has varied from 30 to 125. RESULTS: 50% of the patients had three-vessel and/or left main coronary artery stenosis. Less than 10% of the patients had no significant coronary stenosis or valvular disease at angiography. The total complication rate was 4.8%. Nine patients (1.1%) had myocardial infarction or stroke with sequelae; three patients died (0.4%). INTERPRETATION: Our complication rates are higher than those from other larger hospitals in Norway and internationally, but they have decreased over time. The high percentage of serious coronary pathology among our patients may have contributed significantly to the complication rate. In addition, the annual number of exams may have been too low to give sufficient operator training. The results require a continuing evaluation of our practice.