complement c5a

Summary

Summary: The minor fragment formed when C5 convertase cleaves C5 into C5a and COMPLEMENT C5B. C5a is a 74-amino-acid glycopeptide with a carboxy-terminal ARGININE that is crucial for its spasmogenic activity. Of all the complement-derived anaphylatoxins, C5a is the most potent in mediating immediate hypersensitivity (HYPERSENSITIVITY, IMMEDIATE), smooth MUSCLE CONTRACTION; HISTAMINE RELEASE; and migration of LEUKOCYTES to site of INFLAMMATION.

Top Publications

  1. Humayun S, Gohar M, Volkening K, Moisse K, Leystra Lantz C, Mepham J, et al. The complement factor C5a receptor is upregulated in NFL-/- mouse motor neurons. J Neuroimmunol. 2009;210:52-62 pubmed publisher
    ..C5aR expression was increased in motor neurons in ALS. This data suggests that the early upregulation of C5aR may contribute to motor neuron damage that potentiates excitotoxicity in ALS. ..
  2. Ward P. Functions of C5a receptors. J Mol Med (Berl). 2009;87:375-8 pubmed publisher
    ..Better definition of C5L2 is needed if its in vivo blockade, along with C5aR, is to be considered in complement-dependent inflammatory diseases. ..
  3. Hezmee M, Shiels I, Rolfe B, Mills P. Complement C5a: impact on the field of veterinary medicine. Vet J. 2012;192:264-71 pubmed publisher
    b>Complement C5a is a pro-inflammatory polypeptide produced during activation of the complement cascade in response to foreign antigens or tissue damage secondary to physical or chemical injury...
  4. Kambas K, Markiewski M, Pneumatikos I, Rafail S, Theodorou V, Konstantonis D, et al. C5a and TNF-alpha up-regulate the expression of tissue factor in intra-alveolar neutrophils of patients with the acute respiratory distress syndrome. J Immunol. 2008;180:7368-75 pubmed
    ..This cross-talk between inflammatory mediators and the induction of TF expression in intra-alveolar neutrophils may be a potential target for novel therapeutic strategies to limit ARDS-associated disturbances of coagulation. ..
  5. Hegde G, Meyers Clark E, Joshi S, Sanderson S. A conformationally-biased, response-selective agonist of C5a acts as a molecular adjuvant by modulating antigen processing and presentation activities of human dendritic cells. Int Immunopharmacol. 2008;8:819-27 pubmed publisher
  6. von Köckritz Blickwede M, Konrad S, Foster S, Gessner J, Medina E. Protective role of complement C5a in an experimental model of Staphylococcus aureus bacteremia. J Innate Immun. 2010;2:87-92 pubmed publisher
    ..Here, we have examined the role of complement anaphylatoxin C5a in an experimental model of Staphylococcus aureus bacteremia. Our data provide compelling evidence for a protective role of C5a during staphylococcal bloodstream infection. ..
  7. Haas P, van Strijp J. Anaphylatoxins: their role in bacterial infection and inflammation. Immunol Res. 2007;37:161-75 pubmed
  8. Yuan J, Gou S, Huang J, Hao J, Chen M, Zhao M. C5a and its receptors in human anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Arthritis Res Ther. 2012;14:R140 pubmed publisher
    ..The level of renal CD88 expression could reflect the disease severity of ANCA-associated glomerulonephritis. CD88 expression was downregulated, and C5L2 was upregulated in ANCA-associated glomerulonephritis. ..
  9. Burk A, Martin M, Flierl M, Rittirsch D, Helm M, Lampl L, et al. Early complementopathy after multiple injuries in humans. Shock. 2012;37:348-54 pubmed publisher
    ..These events may participate in the impairment of the innate immune response observed after severe trauma. ..

More Information

Publications62

  1. Lalli P, Strainic M, Yang M, Lin F, Medof M, Heeger P. Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis. Blood. 2008;112:1759-66 pubmed publisher
    ..The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death. ..
  2. Gou S, Yuan J, Chen M, Yu F, Zhao M. Circulating complement activation in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Kidney Int. 2013;83:129-37 pubmed publisher
    ..Thus, systemic activation of complement by the alternative pathway takes place in human AAV. Circulating Bb might be a useful biomarker in assessing disease activity of AAV. ..
  3. Mukherjee P, Thomas S, Pasinetti G. Complement anaphylatoxin C5a neuroprotects through regulation of glutamate receptor subunit 2 in vitro and in vivo. J Neuroinflammation. 2008;5:5 pubmed publisher
    ..Our results suggest that C5a protects against apoptotic pathways in neurons in vitro and in vivo through regulation of GluR2 receptor expression. Complement C5a neuroprotects through regulation of GluR2 receptor subunit.
  4. Bestebroer J, Aerts P, Rooijakkers S, Pandey M, K hl J, Van Strijp J, et al. Functional basis for complement evasion by staphylococcal superantigen-like 7. Cell Microbiol. 2010;12:1506-16 pubmed publisher
    ..Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a-driven influx of neutrophils in mouse peritoneum...
  5. Monk P, Scola A, Madala P, Fairlie D. Function, structure and therapeutic potential of complement C5a receptors. Br J Pharmacol. 2007;152:429-48 pubmed
    ..This review will highlight major developments in C5a receptor research that support C5aR as an important therapeutic target. The intriguing possibilities raised by the existence of a non-signalling C5a receptor are also discussed. ..
  6. Flierl M, Schreiber H, Huber Lang M. The role of complement, C5a and its receptors in sepsis and multiorgan dysfunction syndrome. J Invest Surg. 2006;19:255-65 pubmed
    ..In this review, we describe our present understanding of the role of complement in the inflammatory response during sepsis and MODS. ..
  7. Isfort K, Ebert F, Bornhorst J, Sargin S, Kardakaris R, Pasparakis M, et al. Real-time imaging reveals that P2Y2 and P2Y12 receptor agonists are not chemoattractants and macrophage chemotaxis to complement C5a is phosphatidylinositol 3-kinase (PI3K)- and p38 mitogen-activated protein kinase (MAPK)-independent. J Biol Chem. 2011;286:44776-87 pubmed publisher
    ..is implicated as an amplifier mechanism for chemotactic navigation to end-target chemoattractants, such as complement C5a. Here we show using real-time chemotaxis assays that mouse peritoneal macrophages do not directionally migrate ..
  8. Morgan E, Thoman M, Sanderson S, Phillips J. A novel adjuvant for vaccine development in the aged. Vaccine. 2010;28:8275-9 pubmed publisher
    ..These results indicate that EP67 induces humoral immunity in aged mice not obtainable with alum and CpG. These results support the use of EP67 as a potential vaccine adjuvant suited to the elderly. ..
  9. Ignatius A, Schoengraf P, Kreja L, Liedert A, Recknagel S, Kandert S, et al. Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL-1?. J Cell Biochem. 2011;112:2594-605 pubmed publisher
  10. Manthey H, Woodruff T, Taylor S, Monk P. Complement component 5a (C5a). Int J Biochem Cell Biol. 2009;41:2114-7 pubmed publisher
    ..Thus, C5a has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C5a inhibition an attractive therapeutic strategy. ..
  11. Niederbichler A, Hoesel L, Westfall M, Gao H, Ipaktchi K, Sun L, et al. An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction. J Exp Med. 2006;203:53-61 pubmed
    ..These data suggest that CLP induces C5aR on cardiomyocytes and that in vivo generation of C5a causes C5a-C5aR interaction, causing dysfunction of cardiomyocytes, resulting in compromise of cardiac performance. ..
  12. Woodruff T, Ager R, Tenner A, Noakes P, Taylor S. The role of the complement system and the activation fragment C5a in the central nervous system. Neuromolecular Med. 2010;12:179-92 pubmed publisher
    ..The possible overarching role for C5a in diseases of the CNS is reviewed, and the therapeutic potential of blocking C5a/CD88 interaction is evaluated. ..
  13. Strainic M, Shevach E, An F, Lin F, Medof M. Absence of signaling into CD4? cells via C3aR and C5aR enables autoinductive TGF-?1 signaling and induction of Foxp3? regulatory T cells. Nat Immunol. 2013;14:162-71 pubmed publisher
    ..The resulting iT(reg) cells exerted robust suppression, had enhanced stability and suppressed ongoing autoimmune disease. Antagonism of C3aR and C5aR can also induce functional human iT(reg) cells. ..
  14. Reiman R, Campos Torres A, Martin B, Ting J, Campbell I, Barnum S. Expression of C5a in the brain does not exacerbate experimental autoimmune encephalomyelitis. Neurosci Lett. 2005;390:134-8 pubmed
    ..These results demonstrate that C5a, despite it is pro-inflammatory functions, is not critical to the development and progression of EAE. ..
  15. Sheen T, Cavaco C, Ebrahimi C, Thoman M, Sanderson S, Morgan E, et al. Control of methicillin resistant Staphylococcus aureus infection utilizing a novel immunostimulatory peptide. Vaccine. 2011;30:9-13 pubmed publisher
    ..These results suggest that EP67 may serve as a novel immunotherapeutic for prevention and treatment of CA-MRSA dermal infection. ..
  16. Nozaki M, Raisler B, Sakurai E, Sarma J, Barnum S, Lambris J, et al. Drusen complement components C3a and C5a promote choroidal neovascularization. Proc Natl Acad Sci U S A. 2006;103:2328-33 pubmed
    ..Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD...
  17. Gressner O, Koch A, Sanson E, Trautwein C, Tacke F. High C5a levels are associated with increased mortality in sepsis patients--no enhancing effect by actin-free Gc-globulin. Clin Biochem. 2008;41:974-80 pubmed publisher
    ..However, different to pre-existing in vitro data, a clinically relevant interaction between actin-free Gc-globulin and C5a in terms of prognosis in severe inflammatory conditions is not given. ..
  18. Ingersoll S, Martin C, Barnum S, Martin B. CNS-specific expression of C3a and C5a exacerbate demyelination severity in the cuprizone model. Mol Immunol. 2010;48:219-30 pubmed publisher
    ..Overall, our findings show that although C3a and C5a production in the brain play a negative role during demyelination, these proteins may aid in remyelination. ..
  19. Banda N, Levitt B, Wood A, Takahashi K, Stahl G, Holers V, et al. Complement activation pathways in murine immune complex-induced arthritis and in C3a and C5a generation in vitro. Clin Exp Immunol. 2010;159:100-8 pubmed publisher
    ..We conclude that neither the CP nor LP alone is capable of mediating CAIA in vivo and that mouse sera exhibits a high level of IgG-induced C5a generation in vitro through either the CP or AP. ..
  20. Nishimura T, Myles T, Piliponsky A, Piliposky A, Kao P, Berry G, et al. Thrombin-activatable procarboxypeptidase B regulates activated complement C5a in vivo. Blood. 2007;109:1992-7 pubmed
    ..Its activation, along with protein C, by the endothelial thrombin-TM complex represents a homeostatic response to counteract the inflammatory mediators generated at the site of vascular injury. ..
  21. Lee H, Whitfeld P, Mackay C. Receptors for complement C5a. The importance of C5aR and the enigmatic role of C5L2. Immunol Cell Biol. 2008;86:153-60 pubmed publisher
    ..The development of effective mAbs to human C5aR is an alternative approach to drug development, for this highly attractive target. ..
  22. Lee H, Wu W, Wysoczynski M, Liu R, Zuba Surma E, Kucia M, et al. Impaired mobilization of hematopoietic stem/progenitor cells in C5-deficient mice supports the pivotal involvement of innate immunity in this process and reveals novel promobilization effects of granulocytes. Leukemia. 2009;23:2052-62 pubmed publisher
    ..In conclusion, our data reveal the underappreciated central role of innate immunity in mobilization, in which C5 cleavage fragments through granulocytes orchestrate this process...
  23. Raby A, Holst B, Davies J, Colmont C, Laumonnier Y, Coles B, et al. TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2. Eur J Immunol. 2011;41:2741-52 pubmed publisher
    ..Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation. ..
  24. Hashimoto M, Hirota K, Yoshitomi H, Maeda S, Teradaira S, Akizuki S, et al. Complement drives Th17 cell differentiation and triggers autoimmune arthritis. J Exp Med. 2010;207:1135-43 pubmed publisher
    ..Blockade of C5aR may thus be beneficial for controlling Th17-mediated inflammation and autoimmune disease. ..
  25. Li K, Fazekasova H, Wang N, Peng Q, Sacks S, Lombardi G, et al. Functional modulation of human monocytes derived DCs by anaphylatoxins C3a and C5a. Immunobiology. 2012;217:65-73 pubmed publisher
    ..Complement offers a potential route to modulate human DC function and regulate T cell mediated immunity. ..
  26. Flierl M, Rittirsch D, Chen A, Nadeau B, Day D, Sarma J, et al. The complement anaphylatoxin C5a induces apoptosis in adrenomedullary cells during experimental sepsis. PLoS ONE. 2008;3:e2560 pubmed publisher
    ..Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis. ..
  27. Patel S, Berghout J, Lovegrove F, Ayi K, Conroy A, Serghides L, et al. C5 deficiency and C5a or C5aR blockade protects against cerebral malaria. J Exp Med. 2008;205:1133-43 pubmed publisher
    ..falciparum glycosylphosphatidylinositol and C5aR blockade abrogated these amplified responses. These data provide evidence implicating C5/C5a in the pathogenesis of CM...
  28. Chen N, Mirtsos C, Suh D, Lu Y, Lin W, McKerlie C, et al. C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a. Nature. 2007;446:203-7 pubmed
    ..Our data indicate that C5L2 can function as a positive modulator for both C5a- and C3a-anaphylatoxin-induced responses. ..
  29. Kim G, Mocco J, Hahn D, Kellner C, Komotar R, Ducruet A, et al. Protective effect of C5a receptor inhibition after murine reperfused stroke. Neurosurgery. 2008;63:122-5; discussion 125-6 pubmed publisher
    ..These findings demonstrate that modulation of C5a receptor activity significantly alters the degree of neurological damage after experimental reperfused stroke. ..
  30. Xu R, Wang R, Han G, Wang J, Chen G, Wang L, et al. Complement C5a regulates IL-17 by affecting the crosstalk between DC and gammadelta T cells in CLP-induced sepsis. Eur J Immunol. 2010;40:1079-88 pubmed publisher
    ..These results imply that C5a affects the crosstalk between DC and gammadelta T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL-17. ..
  31. Mullaly S, Kubes P. The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands. J Immunol. 2006;177:8154-63 pubmed
    ..aureus-mediated immune responses. ..
  32. Manthey H, Thomas A, Shiels I, Zernecke A, Woodruff T, Rolfe B, et al. Complement C5a inhibition reduces atherosclerosis in ApoE-/- mice. FASEB J. 2011;25:2447-55 pubmed publisher
    The complement C5a receptor, CD88, is present on many of the cells found within human atherosclerotic plaques, but little is known about the role of C5a in atherogenesis...
  33. Sakuma M, Morooka T, Wang Y, Shi C, Croce K, Gao H, et al. The intrinsic complement regulator decay-accelerating factor modulates the biological response to vascular injury. Arterioscler Thromb Vasc Biol. 2010;30:1196-202 pubmed publisher
    ..Targeting the C3a and C5a receptors may be useful for the prevention of neointimal hyperplasia. ..
  34. Liu J, Lin F, Strainic M, An F, Miller R, Altuntas C, et al. IFN-gamma and IL-17 production in experimental autoimmune encephalomyelitis depends on local APC-T cell complement production. J Immunol. 2008;180:5882-9 pubmed
  35. Crane J, Buller K. Systemic blockade of complement C5a receptors reduces lipopolysacharride-induced responses in the paraventricular nucleus and the central amygdala. Neurosci Lett. 2007;424:10-5 pubmed
    ..Our findings demonstrate that C5a may have a role in the activation of the HPA axis in response to systemic LPS. ..
  36. Schreiber A, Xiao H, Jennette J, Schneider W, Luft F, Kettritz R. C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis. J Am Soc Nephrol. 2009;20:289-98 pubmed publisher
    ..05). In summary, C5a and the neutrophil C5aR may compose an amplification loop for ANCA-mediated neutrophil activation. The C5aR may provide a new therapeutic target for ANCA-induced necrotizing crescentic glomerulonephritis. ..
  37. Gerard N, Lu B, Liu P, Craig S, Fujiwara Y, Okinaga S, et al. An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2. J Biol Chem. 2005;280:39677-80 pubmed
    ..Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease. ..
  38. Johswich K, Martin M, Thalmann J, Rheinheimer C, Monk P, Klos A. Ligand specificity of the anaphylatoxin C5L2 receptor and its regulation on myeloid and epithelial cell lines. J Biol Chem. 2006;281:39088-95 pubmed
    ..Thus, this receptor is unlikely to be directly involved in lipid metabolism. Instead, the identification of stimuli modifying C5L2 expression indicates that C5L2 is a highly regulated scavenger receptor for C5a and C5a-des-Arg(74). ..
  39. Moulton R, Mashruwala M, Smith A, Lindsey D, Wetsel R, Haviland D, et al. Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice. J Leukoc Biol. 2007;82:956-67 pubmed
    ..b>Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production...
  40. Peng Q, Li K, Wang N, Li Q, Asgari E, Lu B, et al. Dendritic cell function in allostimulation is modulated by C5aR signaling. J Immunol. 2009;183:6058-68 pubmed publisher
    ..It also suggests that NF-kappaB signaling induced by down-regulation of cAMP/ protein kinase A pathway and up-regulation of PI3K/AKT pathway following C5a stimulation may contribute to up-regulation of DC function. ..
  41. Schraufstatter I, DiScipio R, Zhao M, Khaldoyanidi S. C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J Immunol. 2009;182:3827-36 pubmed publisher
    ..These results suggest that the anaphylatoxins C3a and C5a present in injured tissues contribute to the recruitment of MSCs and regulation of their behavior. ..
  42. Hao J, Meng L, Xu P, Chen M, Zhao M. p38MAPK, ERK and PI3K signaling pathways are involved in C5a-primed neutrophils for ANCA-mediated activation. PLoS ONE. 2012;7:e38317 pubmed publisher
    ..01, P<0.05, P<0.01 and P<0.01), respectively. Activation of p38MAPK, ERK and PI3K are important steps in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA. ..
  43. Woodruff T, Costantini K, Crane J, Atkin J, Monk P, Taylor S, et al. The complement factor C5a contributes to pathology in a rat model of amyotrophic lateral sclerosis. J Immunol. 2008;181:8727-34 pubmed
    ..This study provides the first demonstration of an involvement of C5a in an ALS model and suggests that inhibitors of complement activation could be beneficial in the treatment of this neurodegenerative disease. ..
  44. Le Roux S, Pepper R, Dufay A, Néel M, Meffray E, Lamandé N, et al. Elevated soluble Flt1 inhibits endothelial repair in PR3-ANCA-associated vasculitis. J Am Soc Nephrol. 2012;23:155-64 pubmed publisher
    ..In summary, these data suggest that anti-PR3 antibodies, and to a much lesser extent anti-MPO antibodies, increase sFlt1 during acute ANCA-associated vasculitis, leading to an antiangiogenic state that hinders endothelial repair. ..
  45. Vieyra M, Leisman S, Raedler H, Kwan W, Yang M, Strainic M, et al. Complement regulates CD4 T-cell help to CD8 T cells required for murine allograft rejection. Am J Pathol. 2011;179:766-74 pubmed publisher
  46. Zhang X, Lewkowich I, Köhl G, Clark J, Wills Karp M, Köhl J. A protective role for C5a in the development of allergic asthma associated with altered levels of B7-H1 and B7-DC on plasmacytoid dendritic cells. J Immunol. 2009;182:5123-30 pubmed publisher
    ..Ex vivo targeting of B7-H1 and B7-DC increased Th2 cytokine production from T cells of wild-type but not of C5aR-targeted mice, suggesting a protective role for C5a through regulation of B7 molecule expression on plasmacytoid DCs. ..
  47. Markiewski M, DeAngelis R, Benencia F, Ricklin Lichtsteiner S, Koutoulaki A, Gerard C, et al. Modulation of the antitumor immune response by complement. Nat Immunol. 2008;9:1225-35 pubmed publisher
    ..Here we show that the generation of complement C5a in a tumor microenvironment enhanced tumor growth by suppressing the antitumor CD8(+) T cell-mediated response...
  48. Griffin R, Costigan M, Brenner G, Ma C, Scholz J, Moss A, et al. Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity. J Neurosci. 2007;27:8699-708 pubmed
    ..We conclude that induction of the complement cascade in spinal cord microglia after peripheral nerve injury contributes to neuropathic pain through the release and action of the C5a anaphylatoxin peptide. ..
  49. Khan M, Maasch C, Vater A, Klussmann S, Morser J, Leung L, et al. Targeting complement component 5a promotes vascular integrity and limits airway remodeling. Proc Natl Acad Sci U S A. 2013;110:6061-6 pubmed publisher
    ..As C3 inhibitors enter the clinics, the simultaneous targeting of this thrombin-mediated complement activation pathway and/or C5a itself may confer significant clinical benefit. ..
  50. Peng T, Hao L, Madri J, Su X, Elias J, Stahl G, et al. Role of C5 in the development of airway inflammation, airway hyperresponsiveness, and ongoing airway response. J Clin Invest. 2005;115:1590-600 pubmed
    ..This study suggests that targeting C5 is a potential clinical approach for treating patients with asthma. ..
  51. Strainic M, Liu J, Huang D, An F, Lalli P, Muqim N, et al. Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells. Immunity. 2008;28:425-35 pubmed publisher
    ..These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability, and their amplification by cognate APC partners thus is critical to T cell costimulation. ..
  52. Huber Lang M, Sarma J, Zetoune F, Rittirsch D, Neff T, McGuire S, et al. Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med. 2006;12:682-7 pubmed
    ..These data suggest that, in the genetic absence of C3, thrombin substitutes for the C3-dependent C5 convertase. This linkage between the complement and coagulation pathways may represent a new pathway of complement activation. ..
  53. Oskeritzian C, Zhao W, Min H, Xia H, Pozez A, Kiev J, et al. Surface CD88 functionally distinguishes the MCTC from the MCT type of human lung mast cell. J Allergy Clin Immunol. 2005;115:1162-8 pubmed
    ..MC(T) and MC(TC) types of human mast cells (MCs) are distinguished from one another on the basis of the protease compositions of their secretory granules, but their functional and developmental relationships have been uncertain...