Synthesis of Complement Components C5, C6, C7, C8 and C9 in Vitro by Human Monocytes and Assembly of the Terminal Complement Complex

1986 ◽  
Vol 24 (4) ◽  
pp. 421-428 ◽  
Author(s):  
G. HETLAND ◽  
E. JOHNSON ◽  
R. J. FALK ◽  
T. ESKELAND
Keyword(s):  
Human Monocytes ◽  
Complement Components ◽  
Terminal Complement Complex ◽  
Terminal Complement

The Alternate Complement Pathway in Thrombotic Thrombocytopenic Purpura

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3342-3342
Author(s):  
John C. Chapin ◽  
Katrina Piskorski ◽  
Stephen J Eyler ◽  
Richard J. H. Smith ◽  
Jeffrey Laurence
Keyword(s):  
Complement Factor ◽  
Adamts13 Activity ◽  
Complement Pathway ◽  
Regulatory Factor ◽  
Complement Components ◽  
Thrombocytopenic Purpura ◽  
Susceptibility Factor ◽  
Alternate Complement Pathway ◽  
Terminal Complement

Abstract Abstract 3342 The thrombotic microangiopathies (TMA) thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS) involve progressive microvascular thrombi, endothelial cell (EC) injury, vascular ischemia, and severe end-organ damage. Acquired TTP is often associated with autoantibody-mediated suppression of the ADAMTS13 vWF cleaving protease, causing vWF multimer accumulation and platelet aggregation. aHUS is associated with dysregulation of the alternate complement pathway through mutation in and/or autoantibodies against, complement regulatory proteins. It is responsive to the anti-C5 mAb eculizumab. TTP, by contrast, usually responds to plasma exchange, but in the refractory setting there are few effective treatments. We hypothesized that dysregulation of the alternate complement pathway represents a susceptibility factor for EC injury in at least a subset of TTP patients. Our objective was to identify the degree of complement dysregulation in acute TTP vs. other TMAs in vivo, and correlate these data with (1) the ability of plasma from acute TTP and aHUS patients to induce apoptotic injury in primary human microvascular EC in vitro and (2) the potential of eculizumab to block this injury. Plasmas from acute TMA patients (TTP n=12, malignancy-associated aHUS n=6, ticlopidine-associated TTP n=4, systemic Degos disease n=1), and healthy controls (n=4) were collected at time of presentation. Samples were assayed for terminal complement components sC5b-9 (MAC, membrane attack complex) and C5a by ELISA. Genomic DNA was isolated from these plasmas and amplified by standard DNA PCR, followed by semi-nested PCR using primers designed around the exon sequences of complement factor H (CFH), complement factor I (CFI) and MCP (CD46) known to be mutated in 60–80% of aHUS cases. Amplicons were sequenced and correlated with a database of previously reported mutations and SNPs with varying degrees of functional significance in the complement regulatory pathway. In our in vitro model for plasma-mediated EC injury, primary human dermal microvascular ECs were starved in medium lacking EC growth factors and then incubated for 18–24 hours with 1–2% plasma (v/v) in the presence or absence of pharmacologic levels of anti-C5 mAb (100–250μg/ml). Apoptosis was assessed by ELISA-based quantification of cytoplasmic histone-associated DNA fragments from cell lysate and propidium iodide labeling with construction of DNA histograms and assessment of A0 peaks by flow cytometry. We found significantly elevated plasma levels of C5a in all subsets of patients with TMAs compared to control plasma (42.8 ng/ml +/− 6.2 vs. 32 ng/ml +/− 6.8; p=0.014). We also found markedly elevated levels sC5b-9 in these TMAs compared to controls (1852.0 ng/ml +/− 1169.8 vs 598.8 +/− 338.7; p=0.012). Little variation was seen in TTP vs. aHUS and other TMAs, regardless of ADAMTS13 status. Complement mutations in CFH and CFI were identified in 14 (66.7%) of TMA patients: 41.6% TTP, 60% malignancy-aHUS, 100% ticlopidine TTP. In terms of interference with TMA plasma-induced MVEC apoptosis in vitro, EC injury was blocked by anti-C5 mAb eculizumab in 8 of 19 cases (5 TTP, 2 aHUS, 1 Degos). Correlation of sensitivity to plasma-mediated EC apoptosis and blockade with eculizumab with levels of terminal complement components, presence of complement regulatory factor mutations, levels of ADAMTS13 activity, and anti-ADAMTS13 antibody titers are underway. We conclude that dysregulation of the alternate complement pathway may represent a susceptibility factor in the pathophysiology of many TMAs, not only aHUS. Blockade of C5 may offer a therapeutic avenue for some patients with refractory TTP. Indeed, in a recent report our group noted the rescue of a patient with classic TTP, including ADAMTS13 activity <5% and anti-ADAMTS13 IgG, refractory to plasma exchange and a variety of immune suppressive regimens, utilizing eculizumab (Chapin J, et al. Brit J Hematol, 2012). Defining this subset, and the potential for clinical response to anti-C5 therapy, will involve exploration of other complement and complement regulatory factor mutations and autoantibodies. Disclosures: Off Label Use: Eculizumab is not FDA approved for use in the treatment of TTP. Laurence:Alexion Pharmaceuticals: Consultancy, Speakers Bureau.

scholarly journals A systems approach to inflammation identifies therapeutic targets in SARS-CoV-2 infection

2020 ◽  
Author(s):  
Frank L. van de Veerdonk ◽  
Nico A.F. Janssen ◽  
Inge Grondman ◽  
Aline H. de Nooijer ◽  
Valerie A.C.M. Koeken ◽  
...  
Keyword(s):  
Systems Approach ◽  
Plasma Concentrations ◽  
Severe Pneumonia ◽  
Targeted Proteomics ◽  
Complement Components ◽  
Icu Patients ◽  
Terminal Complement Complex ◽  
Multicenter Observational Study ◽  
Tnf Α ◽  
Terminal Complement

AbstractBackgroundInfection with SARS-CoV-2 manifests itself as a mild respiratory tract infection in the majority of individuals, which progresses to a severe pneumonia and acute respiratory distress syndrome (ARDS) in 10-15% of patients. Inflammation plays a crucial role in the pathogenesis of ARDS, with immune dysregulation in severe COVID-19 leading to a hyperinflammatory response. A comprehensive understanding of the inflammatory process in COVID-19 is lacking.MethodsIn this prospective, multicenter observational study, patients with PCR-proven or clinically presumed COVID-19 admitted to the intensive care unit (ICU) or clinical wards were included. Demographic and clinical data were obtained and plasma was serially collected. Concentrations of IL-6, TNF-α, complement components C3a, C3c and the terminal complement complex (TCC) were determined in plasma by ELISA. Additionally, 269 circulating biomarkers were assessed using targeted proteomics. Results were compared between ICU and non ICU patients.FindingsA total of 119 (38 ICU and 91 non ICU) patients were included. IL-6 plasma concentrations were elevated in COVID-19 (ICU vs. non ICU, median 174.5 pg/ml [IQR 94.5-376.3] vs. 40.0 pg/ml [16.5-81.0]), whereas TNF-α concentrations were relatively low and not different between ICU and non ICU patients (median 24.0 pg/ml [IQR 16.5-33.5] and 21.5 pg/ml [IQR 16.0-33.5], respectively). C3a and terminal complement complex (TCC) concentrations were significantly higher in ICU vs. non ICU patients (median 556.0 ng/ml [IQR 333.3-712.5]) vs. 266.5 ng/ml [IQR 191.5-384.0] for C3a and 4506 mAU/ml [IQR 3661-6595] vs. 3582 mAU/ml [IQR 2947-4300] for TCC) on the first day of blood sampling. Targeted proteomics demonstrated that IL-6 (logFC 2.2), several chemokines and hepatocyte growth factor (logFC 1.4) were significantly upregulated in ICU vs. non ICU patients. In contrast, stem cell factor was significantly downregulated (logFC −1.3) in ICU vs. non ICU patients, as were DPP4 (logFC −0.4) and protein C inhibitor (log FC −1.0), the latter two factors also being involved in the regulation of the kinin-kallikrein pathway. Unsupervised clustering pointed towards a homogeneous pathogenetic mechanism in the majority of patients infected with SARS-CoV-2, with patient clustering mainly based on disease severity.InterpretationWe identified important pathways involved in dysregulation of inflammation in patients with severe COVID-19, including the IL-6, complement system and kinin-kallikrein pathways. Our findings may aid the development of new approaches to host-directed therapy.FundingVidi grant (F.L.v.d.V.) and Spinoza grant (M.G.N.) from the Netherlands Organization for Scientific Research, and ERC Advanced Grant (#833247 to M.G.N.).

Effect of Cyclic AMP and Cyclic GMP on Thromboplastin (Factor III) Synthesis in Human Monocytes In Vitro

1983 ◽  
Vol 50 (04) ◽  
pp. 804-809 ◽  
Author(s):  
Torstein Lyberg
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Immune Complexes ◽  
Phosphodiesterase Inhibitors ◽  
Inhibitory Effect ◽  
Intracellular Camp ◽  
Human Monocytes ◽  
Purified Protein Derivative ◽  
A 23187

SummaryHuman monocytes in vitro respond to various agents (immune complexes, lectins, endotoxin, the divalent ionophore A 23187, 12-0-tetradecanoyl-phorbol 13-acetate [TPA], purified protein derivative [PPD] of Bacille Calmette-Guerin) with an increased synthesis of the protein component of thromboplastin. The effect of cyclic AMP and cyclic GMP on this response has been studied. Dibutyryl-cyclic AMP, prostaglandin E1 and the phosphodiesterase inhibitors 3-butyl-1-methyl-xanthine (MIX) and rac -4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 201724), separately and in combination have a pronounced inhibitory effect on the response to immune complexes and PPD, and a moderate effect on the response to endotoxin and lectins. The effect on TPA response and on the response to A 23187 was slight. Dibutyryl-cyclic GMP (1 mM) gave a slight inhibition of the TPA arid IC response, but had essentially no effect on the response to other inducers. The intracellular cAMP level increased when monocytes were incubated with IC, TPA or A 23187 followed by a decrease to basal levels within 1-2 hr, whereas lectin (PHA) and PPD did not induce such changes. The cAMP response to endotoxin varied. Stimulation with IC induced an increase in monocyte cGMP levels, whereas the other stimulants did not cause such changes.

Clinical Model: Interferons Activate Human Monocytes to An Eradicative Tumor Cell Level In Vitro

2007 ◽  
Vol 27 (2) ◽  
pp. 157-164 ◽  
Author(s):  
Samuel Baron ◽  
Jessica Hernandez ◽  
Joseph Bekisz ◽  
Joyce Poast ◽  
Neil Goldman ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Human Monocytes ◽  
Cell Level ◽  
Clinical Model

scholarly journals Large-scale reduction of tyrosine kinase activities in human monocytes stimulated in vitro with N. meningitidis

PLoS ONE ◽  
2018 ◽  
Vol 13 (1) ◽  
pp. e0181912 ◽  
Author(s):  
Unni Gopinathan ◽  
Kathrine Røe Redalen ◽  
Anne-Marie Trøseid ◽  
Peter Kierulf ◽  
Petter Brandtzaeg ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Large Scale ◽  
Human Monocytes ◽  
Scale Reduction
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