Publications by authors named "Gerard van Mierlo"

30 Publications

  • Page 1 of 1

Fc Galactosylation Promotes Hexamerization of Human IgG1, Leading to Enhanced Classical Complement Activation.

J Immunol 2021 09 18;207(6):1545-1554. Epub 2021 Aug 18.

Department of Experimental Immunohematology, Sanquin Research and Landsteiner Laboratory, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands;

Human IgG contains one evolutionarily conserved -linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG-Fab regions, allowing neighboring IgG-Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1-Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1-Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1-Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering.
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http://dx.doi.org/10.4049/jimmunol.2100399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428746PMC
September 2021

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Am J Hum Genet 2021 08 13;108(8):1367-1384. Epub 2021 Jul 13.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, 6525EX, the Netherlands; Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525GA, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10), FHR-2 (p = 1.47 × 10), FHR-3 (p = 1.05 × 10) and FHR-4A (p = 1.22 × 10) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10 and p = 2.81 × 10, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387287PMC
August 2021

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Thromb Haemost 2021 Jun 25. Epub 2021 Jun 25.

Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.
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http://dx.doi.org/10.1055/s-0041-1731051DOI Listing
June 2021

The relation between fibrinogen level, neutrophil activity and nucleosomes in the onset of disseminated intravascular coagulation in the critically ill.

J Intern Med 2021 Oct 9;290(4):922-927. Epub 2021 Jul 9.

Laboratory of Experimental Intensive Care and Anesthesiology, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Background: Nucleosomes and neutrophil extracellular traps (NETs) are important in the pathophysiology of disseminated intravascular coagulation (DIC). Fibrinogen, as an acute phase reactant, may be protective by engaging neutrophils. We hypothesize that DIC can occur when NET formation becomes uncontrolled in relation to low fibrinogen levels.

Patients/method: The ratio of both circulating nucleosomes and human neutrophil elastase alpha-1-antitrypsine complexes (HNE-a1ATc) to fibrinogen was correlated to thrombocytopenia, DIC and organ failure in 64 critically ill coagulopathic patients.

Results: A high nucleosome to fibrinogen ratio correlated with thrombocytopenia and organ failure (ρ -0.391, p 0.01 and ρ 0.556, p 0.01, respectively). A high HNE-a1ATc to fibrinogen ratio correlated with thrombocytopenia, DIC and organ failure (ρ -0.418, p 0.01, ρ 0.391, p 0.01 and ρ 0.477, p 0.01 respectively).

Conclusion: These findings support the hypothesis that fibrinogen is protective against DIC by counterbalancing excessive neutrophil activation.
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http://dx.doi.org/10.1111/joim.13346DOI Listing
October 2021

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors.

Clin Transl Immunology 2021 16;10(5):e1285. Epub 2021 May 16.

Department of Immunopathology Sanquin Research Amsterdam The Netherlands.

Objectives: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms.

Methods: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling.

Results: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay.

Conclusion: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.
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http://dx.doi.org/10.1002/cti2.1285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126762PMC
May 2021

Iron-Driven Alterations on Red Blood Cell-Derived Microvesicles Amplify Coagulation during Hemolysis via the Intrinsic Tenase Complex.

Thromb Haemost 2021 May 3. Epub 2021 May 3.

Sanquin Research, Department of Immunopathology, Amsterdam, The Netherlands, and Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Hemolytic disorders characterized by complement-mediated intravascular hemolysis, such as autoimmune hemolytic anemia and paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular hemoglobin, heme, and iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring factor Xa (FXa) and thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during hemolysis stimulate thrombin generation through a mechanism involving FVIII and FIX, the so-called intrinsic tenase complex. Iron scavenging during hemolysis using deferoxamine decreased the ability of the HMVs to enhance thrombin generation. Furthermore, the addition of ferric chloride (FeCl) to plasma propagated thrombin generation in a FVIII- and FIX-dependent manner suggesting that iron positively affects blood coagulation. Phosphatidylserine (PS) blockade using lactadherin and iron chelation using deferoxamine reduced intrinsic tenase activity in a purified system containing HMVs as source of phospholipids confirming that both PS and iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl and HMVs decreased in the presence of ascorbate and glutathione indicating that oxidative stress plays a role in hypercoagulability. Overall, our results provide evidence for the contribution of iron ions derived from hemolytic RBCs to thrombin generation. These findings add to our understanding of the pathogenesis of thrombosis in hemolytic diseases.
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http://dx.doi.org/10.1055/a-1497-9573DOI Listing
May 2021

Biomarkers to predict infection and infection-related complications during chemotherapy-induced neutropenia in acute myeloid leukaemia: a pilot study.

Br J Haematol 2021 06 8;193(5):1008-1012. Epub 2021 Apr 8.

Department of Blood Cell Research, Division Research and Landsteiner Laboratory of Amsterdam UMC, Sanquin Blood Supply, Amsterdam, the Netherlands.

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http://dx.doi.org/10.1111/bjh.17422DOI Listing
June 2021

Sugar Matters: Improving In Vivo Clearance Rate of Highly Glycosylated Recombinant Plasma Proteins for Therapeutic Use.

Pharmaceuticals (Basel) 2021 Jan 11;14(1). Epub 2021 Jan 11.

Department of Immunopathology, Sanquin Research, Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, The Netherlands.

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.
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http://dx.doi.org/10.3390/ph14010054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826800PMC
January 2021

The effects of tidal volume size and driving pressure levels on pulmonary complement activation: an observational study in critically ill patients.

Intensive Care Med Exp 2020 Dec 18;8(Suppl 1):74. Epub 2020 Dec 18.

Department of Intensive Care Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

Background: Mechanical ventilation can induce or even worsen lung injury, at least in part via overdistension caused by too large volumes or too high pressures. The complement system has been suggested to play a causative role in ventilator-induced lung injury.

Aims And Methods: This was a single-center prospective study investigating associations between pulmonary levels of complement activation products and two ventilator settings, tidal volume (V) and driving pressure (ΔP), in critically ill patients under invasive ventilation. A miniature bronchoalveolar lavage (BAL) was performed for determination of pulmonary levels of C5a, C3b/c, and C4b/c. The primary endpoint was the correlation between BAL fluid (BALF) levels of C5a and V and ΔP. Levels of complement activation products were also compared between patients with and without ARDS or with and without pneumonia.

Results: Seventy-two patients were included. Median time from start of invasive ventilation till BAL was 27 [19 to 34] hours. Median V and ΔP before BAL were 6.7 [IQR 6.1 to 7.6] ml/kg predicted bodyweight (PBW) and 15 [IQR 11 to 18] cm HO, respectively. BALF levels of C5a, C3b/c and C4b/c were neither different between patients with or without ARDS, nor between patients with or without pneumonia. BALF levels of C5a, and also C3b/c and C4b/c, did not correlate with V and ΔP. Median BALF levels of C5a, C3b/c, and C4b/c, and the effects of V and ΔP on those levels, were not different between patients with or without ARDS, and in patients with or without pneumonia.

Conclusion: In this cohort of critically ill patients under invasive ventilation, pulmonary levels of complement activation products were independent of the size of V and the level of ΔP. The associations were not different for patients with ARDS or with pneumonia. Pulmonary complement activation does not seem to play a major role in VILI, and not even in lung injury per se, in critically ill patients under invasive ventilation.
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http://dx.doi.org/10.1186/s40635-020-00356-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746430PMC
December 2020

Development of a SARS-CoV-2 Total Antibody Assay and the Dynamics of Antibody Response over Time in Hospitalized and Nonhospitalized Patients with COVID-19.

J Immunol 2020 12 30;205(12):3491-3499. Epub 2020 Oct 30.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory Academic Medical Centre, 1066 CX Amsterdam, the Netherlands;

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients ( = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors ( = 182), PCR-confirmed hospital care workers ( = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 ( = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.
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http://dx.doi.org/10.4049/jimmunol.2000767DOI Listing
December 2020

Unraveling the Effect of a Potentiating Anti-Factor H Antibody on Atypical Hemolytic Uremic Syndrome-Associated Factor H Variants.

J Immunol 2020 10 26;205(7):1778-1786. Epub 2020 Aug 26.

Department of Immunopathology, Sanquin Research, Landsteiner Laboratory, Amsterdam UMC, University of Amsterdam, 1066 CX Amsterdam, the Netherlands;

The complement system plays an important role in our innate immune system. Complement activation results in clearance of pathogens, immune complex, and apoptotic cells. The host is protected from complement-mediated damage by several complement regulators. Factor H (FH) is the most important fluid-phase regulator of the alternative pathway of the complement system. Heterozygous mutations in FH are associated with complement-related diseases such as atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration. We recently described an agonistic anti-FH mAb that can potentiate the regulatory function of FH. This Ab could serve as a potential new drug for aHUS patients and alternative to C5 blockade by eculizumab. However, it is unclear whether this Ab can potentiate FH mutant variants in addition to wild-type (WT) FH. In this study, the functionality and potential of the agonistic Ab in the context of pathogenic aHUS-related FH mutant proteins was investigated. The binding affinity of recombinant WT FH and the FH variants, W1183L, V1197A, R1210C, and G1194D to C3b was increased upon addition of the potentiating Ab and similarly, the decay-accelerating activity of all mutants is increased. The potentiating anti-FH Ab is able to restore the surface regulatory function of most of the tested FH mutants to WT FH levels on a human HAP-1 cell line and on sheep erythrocytes. In conclusion, our potentiating anti-FH is broadly active and able to enhance both WT FH function as well as most aHUS-associated FH variants tested in this study.
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http://dx.doi.org/10.4049/jimmunol.2000368DOI Listing
October 2020

Complement factor H contributes to mortality in humans and mice with bacterial meningitis.

J Neuroinflammation 2019 Dec 28;16(1):279. Epub 2019 Dec 28.

Department of Neurology, Amsterdam UMC, University of Amsterdam, Amsterdam Neuroscience, Amsterdam, the Netherlands.

Background: The complement system is a vital component of the inflammatory response occurring during bacterial meningitis. Blocking the complement system was shown to improve the outcome of experimental pneumococcal meningitis. Complement factor H (FH) is a complement regulatory protein inhibiting alternative pathway activation but is also exploited by the pneumococcus to prevent complement activation on its surface conferring serum resistance.

Methods: In a nationwide prospective cohort study of 1009 episodes with community-acquired bacterial meningitis, we analyzed whether genetic variations in CFH influenced FH cerebrospinal fluid levels and/or disease severity. Subsequently, we analyzed the role of FH in our pneumococcal meningitis mouse model using FH knock-out (Cfh) mice and wild-type (wt) mice. Finally, we tested whether adjuvant treatment with human FH (hFH) improved outcome in a randomized investigator blinded trial in a pneumococcal meningitis mouse model.

Results: We found the major allele (G) of single nucleotide polymorphism in CFH (rs6677604) to be associated with low FH cerebrospinal fluid concentration and increased mortality. In patients and mice with bacterial meningitis, FH concentrations were elevated during disease and Cfh mice with pneumococcal meningitis had increased mortality compared to wild-type mice due to C3 depletion. Adjuvant treatment of wild-type mice with purified human FH led to complement inhibition but also increased bacterial outgrowth which resulted in similar disease outcomes.

Conclusion: Low FH levels contribute to mortality in pneumococcal meningitis but adjuvant treatment with FH at a clinically relevant time point is not beneficial.
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http://dx.doi.org/10.1186/s12974-019-1675-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935240PMC
December 2019

α1-Antichymotrypsin Present in Therapeutic C1-Inhibitor Products Competes with Selectin-Sialyl LewisX Interaction.

Thromb Haemost 2018 Dec 19;118(12):2134-2144. Epub 2018 Nov 19.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory of the AMC, University of Amsterdam, Amsterdam, The Netherlands.

Background:  C1-inhibitor (C1-inh) therapeutics can reduce neutrophil activity in various inflammatory conditions. This 'novel' anti-inflammatory effect of C1-inh is attributed to the tetrasaccharide sialyl Lewis (SLe) present on its glycans. Via SLe, C1-inh is suggested to interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain plasma glycoprotein α1-antichymotrypsin (ACT) as a co-purified protein impurity.

Objective:  This article investigates the contribution of ACT to the effects observed with C1-inh.

Materials And Methods:  We have separated C1-inh and ACT from a therapeutic C1-inh preparation and investigated the influence of these proteins on SLe-selectin interactions in a specific in vitro model, which makes use of rolling of SLe-coated beads on immobilized E-selectin.

Results:  We find that ACT and not C1-inh, shows a clear sialic acid-dependent interference in SLe-selectin interactions, at concentrations present in C1-inh therapeutics. Furthermore, we do not find any evidence of SLe on C1-inh using either Western blotting with anti-SLe antibodies (CSLEX1 and KM93) or by mass spectrometric analysis of glycans. C1-inh reacts weakly to antibody HECA-452, which detects a broad range of selectin ligands, but ACT gives a much stronger signal, suggesting the presence of a selectin ligand on ACT.

Conclusion:  The 'novel' anti-inflammatory effects of C1-inh are unlikely due to SLe on C1-inh and can in fact be due to SLe-like glycans on ACT, present in C1-inh products. In view of our results, it is important to assess the role of ACT in vivo and revisit past studies performed with commercial C1-inh.
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http://dx.doi.org/10.1055/s-0038-1675601DOI Listing
December 2018

Substitution of Mannan-Binding Lectin (MBL)-Deficient Serum With Recombinant MBL Results in the Formation of New MBL/MBL-Associated Serine Protease Complexes.

Front Immunol 2018 27;9:1406. Epub 2018 Jun 27.

Department of Immunopathology, Sanquin Blood Supply, Division Research and Landsteiner Laboratory of the Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands.

The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL-MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.
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http://dx.doi.org/10.3389/fimmu.2018.01406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030254PMC
June 2018

Factor H-Related (FHR)-1 and FHR-2 Form Homo- and Heterodimers, while FHR-5 Circulates Only As Homodimer in Human Plasma.

Front Immunol 2017 18;8:1328. Epub 2017 Oct 18.

Department of Immunopathology, Sanquin Research and Landsteiner Laboratory of the Academic Medical Centre, University of Amsterdam, Amsterdam, Netherlands.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous deletions or compound heterozygous missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed , using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.
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http://dx.doi.org/10.3389/fimmu.2017.01328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651247PMC
October 2017

Human plasma-derived C1 esterase inhibitor concentrate has limited effect on house dust mite-induced allergic lung inflammation in mice.

PLoS One 2017 16;12(10):e0186652. Epub 2017 Oct 16.

Department of Immunopathology, Sanquin Research, Amsterdam, the Netherlands.

C1 esterase inhibitor (C1-INH) can inhibit multiple pathways (complement, contact-kinin, coagulation, and fibrinolysis) that are all implicated in the pathophysiology of asthma. We explored the effect of human plasma-derived C1-INH on allergic lung inflammation in a house dust mite (HDM) induced asthma mouse model by daily administration of C1-INH (15 U) during the challenge phase. NaCl and HDM exposed mice had comparable plasma C1-INH levels, while bronchoalveolar lavage fluid (BALF) levels were increased in HDM exposed mice coinciding with slightly reduced activation of complement (C5a). C1-INH treatment reduced Th2 response and enhanced HDM-specific IgG1. Influx of eosinophils in BALF or lung, pulmonary damage, mucus production, procoagulant response or plasma leakage in BALF was similar in both groups. In conclusion, C1-INH dampens Th2 responses during HDM induced allergic lung inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186652PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643136PMC
November 2017

Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

Cell Biochem Biophys 2016 Dec 28;74(4):545-552. Epub 2016 Sep 28.

Laboratory of Experimental Intensive Care and Anesthesiology (L·E·I·C·A), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.
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http://dx.doi.org/10.1007/s12013-016-0766-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101262PMC
December 2016

Activated protein C inhibits neutrophil migration in allergic asthma: a randomised trial.

Eur Respir J 2015 Dec 17;46(6):1636-44. Epub 2015 Sep 17.

Center of Infection and Immunity Amsterdam, and Center for Experimental and Molecular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Asthma patients show evidence of a procoagulant state in their airways, accompanied by an impaired function of the anticoagulant protein C system. We aimed to study the effect of recombinant human activated protein C (rhAPC) in allergic asthma patients.We conducted a randomised, double-blind, placebo-controlled, proof-of-concept study in house dust mite (HDM) allergic asthma patients. Patients were randomised to receive intravenous rhAPC (24 µg·kg(-1)·h(-1); n=12) or placebo (n=12) for 11 h. 4 h after the start of infusion, a first bronchoscopy was performed to challenge one lung segment with saline (control) and a contralateral segment with a combination of HDM extract and lipopolysaccharide (HDM+LPS), thereby mimicking environmental house dust exposure. A second bronchoscopy was conducted 8 h after intrabronchial challenge to obtain bronchoalveolar lavage fluid (BALF).rhAPC did not influence HDM+LPS induced procoagulant changes in the lung. In contrast, rhAPC reduced BALF leukocyte counts by 43% relative to placebo, caused by an inhibitory effect on neutrophil influx (64% reduction), while leaving eosinophil influx unaltered. rhAPC also reduced neutrophil degranulation products in the airways.Intravenous rhAPC attenuates HDM+LPS-induced neutrophil migration and protein release in allergic asthma patients by an effect that does not rely on coagulation inhibition.
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http://dx.doi.org/10.1183/13993003.00459-2015DOI Listing
December 2015

Increased intra- and extracellular granzyme expression in patients with tuberculosis.

Tuberculosis (Edinb) 2015 Sep 20;95(5):575-80. Epub 2015 Jun 20.

Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; Division of Infectious Diseases, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Electronic address:

Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. Granzymes (gzms) are proteases mainly found in cytotoxic lymphocytes, but also extracellularly. While the role of gzms in target cell death has been widely characterized, considerable evidence points towards broader roles related to infectious and inflammatory responses. To investigate the expression of the gzms in TB, intracellular gzms A, B and K were measured by flow cytometry in lymphocyte populations from peripheral blood mononuclear cells from 18 TB patients and 12 healthy donors from Bangladesh, and extracellular levels of gzmA and B were measured in serum from 58 TB patients and 31 healthy controls. TB patients showed increased expression of gzmA in CD8(+) T, CD4(+) T and CD56(+) T, but not NK, cells, and of gzmB in CD8(+) T cells, when compared to controls. GzmK expression was not altered in TB patients in any lymphocyte subset. The extracellular levels of gzmA and, to a lesser extent, of gzmB, were increased in TB patients, but did not correlate with intracellular gzm expression in lymphocyte subsets. Our results reveal enhanced intra- and extracellular expression of gzmA and B in patients with pulmonary TB, suggesting that gzms are part of the host response to tuberculosis.
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http://dx.doi.org/10.1016/j.tube.2015.05.016DOI Listing
September 2015

HIV infection is associated with elevated nucleosomes in asymptomatic patients and during sepsis or malaria.

J Infect 2015 Aug 3;71(2):266-9. Epub 2015 Apr 3.

Centre of Experimental and Molecular Medicine, Division of Infectious Diseases, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.

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http://dx.doi.org/10.1016/j.jinf.2015.03.008DOI Listing
August 2015

HIV Coinfection Enhances Complement Activation During Sepsis.

J Infect Dis 2015 Aug 5;212(3):474-83. Epub 2015 Feb 5.

Center of Experimental and Molecular Medicine.

Background: Human immunodeficiency virus (HIV)-induced complement activation may play a role in chronic immune activation in patients with HIV infection and influence the complement system during acute illness. We determined the impact of HIV infection on the complement system in patients with asymptomatic HIV infection and HIV-infected patients with sepsis or malaria.

Methods: We performed a prospective observational study of 268 subjects with or without HIV infection who were asymptomatic, were septic, or had malaria. We measured complement activation products (C3bc and C4bc) and native complement proteins (C3 and C4). levels of mannose-binding lectin and C1q-C4 were measured to examine activation of the lectin and classical pathways, respectively.

Results: Asymptomatic HIV infection was associated with increased C4 activation, especially in patients with high HIV loads, and was accompanied by elevated C1q-C4 levels. Similarly, sepsis and malaria resulted in increased C4 activation and elevated C1q-C4 concentrations. HIV coinfection enhanced C4 activation and consumption in patients with sepsis; this effect was not detected in patients with malaria. Mannose-binding lectin deficiency (defined as a mannose-binding lectin level of <500 ng/mL) did not influence complement activation in any group.

Conclusions: HIV activates the complement system, predominantly via the classical pathway, and causes increased C4 activation and consumption during sepsis. HIV-induced complement activation may contribute to tissue injury during chronic infection and acute intercurrent bacterial infections.
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http://dx.doi.org/10.1093/infdis/jiv074DOI Listing
August 2015

Interaction of Bordetella pertussis filamentous hemagglutinin with human TLR2: identification of the TLR2-binding domain.

APMIS 2015 Feb 29;123(2):156-62. Epub 2014 Oct 29.

Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

Filamentous hemagglutinin (FHA) is a major adhesion and virulence factor of Bordetella pertussis and also a main component of acellular pertussis vaccines. Interaction of FHA with different receptors on human epithelial and immune cells facilitates entrance and colonization of bacteria as well as immunomodulation of the host immune response. Three overlapping segments of the FHA gene were cloned in a prokaryotic expression vector and the recombinant proteins were purified. These recombinant fragments along with the native FHA protein were employed to assess their potential Toll-like receptor (TLR) stimulatory effects and to localize the TLR binding region. TLR stimulation was monitored by applying HEK293-Blue cell lines cotransfected with TLR2, 4, or 5 and a NF-κB reporter gene. Culture supernatants were checked for secretion of the reporter gene product and IL-8 as indicators of TLR stimulation. Native FHA was found to strongly stimulate TLR2, but not TLR4 or TLR5 transfected cells. Among recombinant FHA fragments only the fragment spanning amino acid residues 1544-1917 was able to exhibit the TLR2 stimulating property of FHA. Interaction of FHA with TLR2 suggests its involvement in induction of the innate immune system against Bordetella pertussis. The TLR2-binding domain of FHA may contribute to immunoprotection against pertussis infection.
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http://dx.doi.org/10.1111/apm.12332DOI Listing
February 2015

Nucleosomes and neutrophil activation in sickle cell disease painful crisis.

Haematologica 2013 Nov 2;98(11):1797-803. Epub 2013 Aug 2.

Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ(0)-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ(+-)thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome.
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http://dx.doi.org/10.3324/haematol.2013.088021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815182PMC
November 2013

Circulating nucleosomes and neutrophil activation as risk factors for deep vein thrombosis.

Arterioscler Thromb Vasc Biol 2013 Jan 25;33(1):147-51. Epub 2012 Oct 25.

Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

Objective: The formation of neutrophil extracellular traps and the exposure of nucleosomes on these neutrophil extracellular traps contribute to coagulation activation and the propagation of deep vein thrombosis (DVT) in animal models. However, no data are available on the role of neutrophil extracellular traps or nucleosomes in patients with thrombosis.

Methods And Results: We conducted a case-control study, in which levels of circulating nucleosomes and neutrophil elastase-α1-antitrypsin complexes were assessed in plasma from 150 patients with objectified symptomatic DVT (cases) and compared with 195 patients with a clinical suspicion of DVT but in whom DVT was excluded (controls). We explored the association between both nucleosomes and elastase-α1-antitrypsin complexes, and the presence of DVT by calculating the odds ratio with corresponding 95% CIs. Elevated levels of both circulating nucleosomes and elastase-α1-antitrypsin complexes were associated with a 3-fold risk of DVT, and the associations remained similar after adjustment for potential confounders (malignancy, smoking, recent immobilization, recent hospitalization). The risk increased with higher nucleosome and elastase-α1-antitrypsin complex levels, suggesting a dose-dependent relationship among circulating nucleosomes, activated neutrophils, and DVT.

Conclusions: Our study suggests an association among circulating nucleosomes, activated neutrophils, and presence of DVT in humans, which might have implications for treatment and prevention.
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http://dx.doi.org/10.1161/ATVBAHA.112.300498DOI Listing
January 2013

Activated cytotoxic T cells and NK cells in severe sepsis and septic shock and their role in multiple organ dysfunction.

Clin Immunol 2005 Aug;116(2):158-65

Central Hematology Laboratory, Inselspital, Bern, Switzerland.

To evaluate the role of activated cytotoxic cells in patients with severe sepsis (n = 32) or septic shock (n = 8), direct (granzymes A and B) as well as indirect markers (cytokines) for cytotoxic cell activation were measured. Elevated IL-12p40 levels had been detected in 58% of the sepsis patients, whereas only a few had detectable TNF-alpha, IFN-gamma, or IL-12p70 levels. Granzymes A and B levels were elevated in 42.5% and 22.5%, respectively. IL-12p40 inversely correlated with disease severity. Inflammatory parameters (IL-6) and coagulation markers were significantly lower and higher, respectively, in patients with elevated IL-12p40 and granzyme B levels, as compared to those with normal levels. Elevation of granzyme A directly correlated with the increase of apoptotic markers. Activated cytotoxic cells reflected by elevated granzymes A and/or B were found in 50% of our sepsis patients. This group showed a higher mortality and a worse organ function.
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http://dx.doi.org/10.1016/j.clim.2005.03.006DOI Listing
August 2005

Complement activation by necrotic cells in normal plasma environment compares to that by late apoptotic cells and involves predominantly IgM.

Eur J Immunol 2004 Sep;34(9):2609-19

Sanquin Research, CLB, Department of Immunopathology, Amsterdam, The Netherlands.

Necrotic cells are generally considered to stimulate inflammation, whereas apoptotic cells should not. However, apoptotic cells have pro-inflammatory properties since they can activate complement. To what extent this activation compares to that by necrotic cells is not known. We compared complement activation by necrotic cells and apoptotic cells in plasma. Jurkat cells were made apoptotic or necrotic by incubation with etoposide or by heat shock, respectively. Cells incubated in recalcified plasma were tested for C3 and C4 fixation and fluid phase generation of complement activation products. Fixation of C3 and C4 to necrotic cells occurred mainly via the classical pathway, independent from the method of necrosis induction and the cell type. Depletion of IgM from plasma almost completely abrogated complement fixation by necrotic cells, which was restored by supplementation with purified IgM. Complement activation by late apoptotic cells was comparable to that by necrotic cells regarding the extent and dependence on IgM. Moreover, incubation of plasma with necrotic or late apoptotic cells led to the generation of comparable amounts of complement activation products. These results indicate that late apoptotic and necrotic cells employ similar complement activation mechanisms in the plasma environment.
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http://dx.doi.org/10.1002/eji.200425045DOI Listing
September 2004

Rat C-reactive protein activates the autologous complement system.

Immunology 2003 Aug;109(4):564-71

Department of Immunopathology, Sanquin Research, and Laboratory for Experimental and Clinical Immunology, Academical Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Activation of complement is a biological function of human C-reactive protein (hCRP), whereas rat CRP (rCRP) has been claimed to be unable to activate complement. As important biological functions of proteins are probably conserved among species, we re-evaluated, using various ligands, the capability of rCRP to activate complement. The activation of complement by hCRP and rCRP was investigated in solid- and fluid-phase systems. In the solid-phase system, purified CRP was fixed to enzyme-linked immunosorbent assay (ELISA) plates and incubated with human or rat recalcified plasma. Dose-dependent binding of human and rat C3 and C4 was observed to human and rat CRP, respectively. In the fluid-phase system, recalcified rat plasma, which contains about 500 mg/l of CRP, or human plasma supplemented with hCRP, were incubated with lyso-phosphatidylcholine. A dose-dependent activation of complement was observed upon incubation with this ligand, as reflected by the generation of activated C4 as well as of CRP-complement complexes. This activation was, in both cases, inhibited by preincubation of plasma with p-aminophosphorylcholine, a specific inhibitor of the interaction of CRP with its ligands, or by chelation of calcium ions. We conclude that rat CRP, similarly to human CRP, can activate autologous complement. These results support the notion that opsonization of ligands with complement is an important biological function of CRP.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783000PMC
http://dx.doi.org/10.1046/j.1365-2567.2003.01681.xDOI Listing
August 2003

Administration of C1 inhibitor reduces neutrophil activation in patients with sepsis.

Clin Diagn Lab Immunol 2003 Jul;10(4):529-35

Central Hematology Laboratory, Inselspital, Bern, Switzerland.

Forty patients with severe sepsis or septic shock recently received C1 inhibitor. In the present study we studied the effect of C1 inhibitor therapy on circulating elastase-alpha(1)-antitrypsin complex (EA) and lactoferrin (LF) levels in these patients to gain further insight about agonists involved in the activation of neutrophils in human sepsis. Elevated levels of EA and LF were found in 65 and 85% of the septic patients, respectively. Patients with elevated EA levels had higher organ dysfunction scores, higher levels of cytokines, and higher levels of complement activation products than patients with normal EA levels. C1 inhibitor therapy reduced EA as well as complement activation and IL-8 release in the patients with elevated EA on admission. We conclude that neutrophil activation in human sepsis correlates with the severity of organ dysfunction and involves complement and interleukin-8 as agonists. The effect of C1 inhibitor therapy on neutrophils may provide an explanation for the beneficial, although mild, effects of this treatment on organ dysfunction in sepsis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC164269PMC
http://dx.doi.org/10.1128/cdli.10.4.529-535.2003DOI Listing
July 2003

Elevated nucleosome levels in systemic inflammation and sepsis.

Crit Care Med 2003 Jul;31(7):1947-51

University Hospital, Bern, Switzerland.

Objective: Multiple organ dysfunction syndrome is a frequent complication of severe sepsis and septic shock and has a high mortality. We hypothesized that extensive apoptosis of cells might constitute the cellular basis for this complication.

Design: Retrospective study.

Setting: Medical and surgical wards or intensive care units of two university hospitals.

Patients: Fourteen patients with fever, 15 with systemic inflammatory response syndrome, 32 with severe sepsis, and eight with septic shock.

Interventions: None.

Measurements And Main Results: We assessed circulating levels of nucleosomes, specific markers released by cells during the later stages of apoptosis, with a previously described enzyme-linked immunosorbent assay in these 69 patients with fever, systemic inflammatory response syndrome, severe sepsis, or septic shock. Severity of multiple organ dysfunction syndrome was assessed with sepsis scores, and clinical and laboratory variables. Elevated nucleosome levels were found in 64%, 60%, 94%, and 100% of patients with fever, systemic inflammatory response syndrome, severe sepsis, or septic shock, respectively. These levels were significantly higher in patients with septic shock as compared with patients with severe sepsis, systemic inflammatory response syndrome, or fever, and in nonsurvivors as compared with survivors. In patients with advanced multiple organ dysfunction syndrome, nucleosome levels correlated with cytokine plasma levels as well as with variables predictive for outcome.

Conclusions: Patients with severe sepsis and septic shock have elevated plasma levels of nucleosomes. We suggest that apoptosis, probably resulting from exposure of cells to excessive amounts of inflammatory mediators, might by involved in the pathogenesis of multiple organ dysfunction syndrome.
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http://dx.doi.org/10.1097/01.CCM.0000074719.40109.95DOI Listing
July 2003
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